JP2016521549A - Method for regulating N-glycosylation site occupancy of plant-produced glycoproteins and recombinant glycoproteins - Google Patents

Abstract

A process for producing a recombinant glycoprotein in a plant, plant cell or plant cell comprising the step of expressing a nucleic acid sequence encoding a polypeptide in said plant, said plant cell or said plant cell Has an N-glycosylation site of the consensus sequence Asn-X-Ser or Asn-X-Thr, where X is any standard amino acid residue, where Asn of said N-glycosylation site When the residue is assigned to position 0 of the amino acid sequence, the amino acid residue at position (a) +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met, or (b) -1 The above process, wherein the amino acid residue at position is selected from Glu and Asp. [Selection figure] None

Description

  The present invention relates to plants, plant cells and processes for producing recombinant glycoproteins in plant cells, which glycoproteins have a high N-glycan occupancy at the N-glycosylation site. The present invention also relates to a method for controlling the occupancy of N-glycans at N-glycosylation sites of recombinant glycoproteins, specifically IgG monoclonal antibodies, in plants. The present invention also provides recombinant glycoproteins having specific amino acid residues adjacent to the N-glycosylation site of the consensus sequence Asn-X-Ser / Thr.

  N-glycosylation is known to play an important role in determining the biological activity and pharmacokinetic properties of many biopharmaceuticals. N-glycosylation at the asparagine residue 297 of immunoglobulin G (IgG) plays a critical role in antibody stability and immune cell mediated Fc effector function. For example, the presence of a glycan at this conserved glycosylation site in an IgG Fc domain, as well as the glycan structure itself, is critical to promote the interaction between mAb and Fc receptor (FcR). Different forms of glycosylation (eg, different glycan structures) define different effects and have been shown to be beneficial and some harmful to the biological and pharmacokinetic properties of mAbs. ing. For example, defucosylated and glycosylated therapeutic mAbs such as Herceptin produced in CHO cells (Shields, RL, et al., 2002, J. Biol. Chem., 277: 26733-26740) and small De-fucosylated anti-CD30 mAb (Cox, KM et al., 2006, Nat. Biotechnol., 24: 1591-1597) expressed in Lemna minor, an aquatic plant of Fc gamma receptor IIIa (FcγRIIIa) Have approximately 30-50 times greater activity than equivalents with alpha-1,6 (CHO cells) or alpha-1,3 (duckweed) linked fucose residues in terms of the effect of ADCC mediated by It was shown that. Similar results have been reported for rituximab and other mAbs produced in animal and plant cells (Shinkawa, T. et al., 2003, J. Biol. Chem., 278: 3466-3473; Niwa, R .; 2004, Cancer Res., 64: 2127-2133; Zeitlin, L., et al., 2011, Proc. Natl. Acad. Sci. USA, 108: 20690-20694, Gasdaska, JR., Et al., 2012, Mol. 50: 134-141).

  The absence of glycans at the glycosylation site in the Fc region has a detrimental effect on the biological activity and pharmacokinetic properties of recombinant mAbs. Removal of N-glycans significantly attenuates ADCC and CDC of CHO cell-produced mAbs (Jefferis R., 2007, Expert Opin Biol Ther., 7: 1401-13). Unglycosylated cetuximab, an epidermal growth factor receptor inhibitor used to treat metastatic colorectal and head and neck cancers, does not bind to FcγRI or FcγRIIIa, and is a high effector-target cell There is no ADCC activity even at a ratio (Patel, D. et al., 2010, Hum Antibodies, 19: 89-99). Structural studies suggest that N-glycans exert their effects mainly through stabilization of the Fc domain conformation (Mimura, Y. et al., 2000, Mol Immunol., 37: 697-706; Sondermann). , P. et al., 2000, Nature, 406: 267-273; Mimura, Y. et al., 2001, J Biol Chem., 276: 45539-45547). Recent detailed studies have demonstrated that hemiglycosylation does not affect Fab-mediated antigen binding and does not affect neonatal Fc receptor binding. However, the semi-glycosylated mAb-X slightly reduces the thermal stability of the CH2 domain, and more importantly, the half-glycosylated forms include high affinity FcγRI, as well as low affinity FcγRIIA, FcγRIIB. Show significantly reduced binding affinity for all Fc gamma receptors (FcγR), including FcγRIIIA and FcγRIIIB (Ha, S. et al., 2011, Glycobiology, 21: 1087-1091). In the case described above, the reduced binding affinity of hemiglycosylated mAb-X to FcγR results in a 3.5-fold antibody-dependent cytotoxic activity (ADCC) compared to the fully glycosylated equivalent. ). ADCC often plays an important role in the therapeutic efficacy of therapeutic antibodies, but glycosylation status is expected not only to affect antibody quality, but also to affect the biological function of the product. .

  Commercially available therapeutic antibodies show 99-100% Fc glycosylation site occupancy, whereas samples of monoclonal antibodies from transgenic plants often contain significant amounts of non-glycosylated variants. [Karnup, A .; S. Kuppannan, K .; And Young, S .; A. 2007, J. MoI. Chromatogr. B, Analyt. Technol. Biomed. Life Sci. 859: 178-191; Giorno, C .; 2010, “Glycoengineering of Monoclonal Antibodies”, Doctoral Dissertation, University of Konstanz]. Our internal data also revealed that plant-produced mAbs often contain up to 30% non-glycosylated N-glycosylation sites in the Fc domain. Such data shows that the final product produced in the plant has a higher heterogeneity compared to the CHO cell-produced mAb, and the bioactivity and pharmacokinetic properties of the plant-produced mAb May have detrimental effects on

  However, the efficient interaction of effector cells with Fc receptors to elicit the patient's immune system through the enhancement of ADCC and CDC has been found in several therapeutic mAbs, including those for cancer treatment. It may not be critical. Effector cell activation may not be necessary for mAbs used as modulators (agonists or antagonists) of signal transduction. Further, since glycosylation of Fc is necessary to maintain the three-dimensional structure and stability of the monoclonal antibody (Zheng, K., Bantoq, C. and Bayer, R., 2011, Mabs, 3: 568- 576; Mimura, Y. et al., 2000, Mol Immunol., 37: 697-706; Sondermann, P. et al., 2000, Nature, 406: 267-273; Mimura, Y. et al., 2001, J Biol Chem. : 45539-45547), complete removal of glycosylation sites, such as by site-directed mutagenesis, may not be the optimal solution.

  Modulation of glycosylation site occupancy of the Fc region of IgG antibodies is assisted by antibody cocktails targeting immune activators and suppressors (Kojima, T. et al., 2010, J. Immunol., 184). : 5493-5501), or a second treatment that, for example, uses trastuzumab, an antibody that targets tumors, to continuously treat HER2-positive cancer, followed by activation of the host's innate immune system When treating with an antibody agonist (anti-CD137) (Kohrt, HE et al., 2012, J. Clin. Invest., 122: 1066-1075), it may be useful when using combination therapy.

  It is therefore an object of the present invention to provide a process for regulating the occupancy of N-glycosylation sites by glycans in the Fc region of immunoglobulins expressed by plants and other glycoproteins expressed by plants. For one purpose. Also, the occupancy of N-glycosylation sites in the Fc region of immunoglobulins expressed by plants and other glycoproteins expressed by plants is greater than 90% or up to almost 100% or less than 50% It is also an object to provide a process that regulates by glycans at different levels, such as Providing a recombinant glycoprotein such as an immunoglobulin (especially IgG) expressed in plants and having a desired glycan occupancy at the N-glycosylation site, particularly at the N-glycosylation site of the Fc region of IgG, It is one purpose.

  These objectives are achieved by:

(1) A process for producing a recombinant glycoprotein in a plant, plant cell or plant cell, the method comprising expressing a nucleic acid encoding the polypeptide in the plant, plant cell or plant cell, Has an N-glycosylation site of the consensus sequence Asn-X-Ser or Asn-X-Thr, where X is any standard amino acid residue, where Asn of said N-glycosylation site When the residue is assigned to position 0 of the amino acid sequence,
(A) the amino acid residue at position +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met; or (b) the amino acid residue at position -1 is selected from Glu and Asp;
As a result, the process described above, wherein the recombinant glycoprotein can be produced.

(2) A nucleic acid encoding a polypeptide comprising CH2 of an IgG heavy chain constant segment having the N-glycosylation site of the consensus sequence Asn-X-Ser or Asn-X-Thr is used as the plant, plant cell or plant cell Wherein X is any standard amino acid residue, wherein the Asn residue of the N-glycosylation site is assigned to position 0 of the amino acid sequence,
(A) the amino acid residue at position +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met, or (b) the amino acid residue at position -1 is selected from Glu and Asp ,
The process according to item 1.

  (3) The process according to item 1 or 2, wherein in the plant cell, the expressed polypeptide is N-glycosylated by post-translational modification at the N-glycosylation site to form a recombinant glycoprotein. .

  (4) N-glycosylation is performed at the N-glycosylation site by the native glycosylation mechanism of the plant to add a plant-type N-glycan to the N-glycosylation site, or human type or 4. The process of item 3, wherein the plant or plant cell has an N-glycosylation mechanism that is genetically engineered, such as to produce humanized N-glycans with N-glycosylation.

  (5) Items 1 to 4 wherein the polypeptide encoded by the nucleic acid comprises an IgG heavy chain constant region, and preferably the polypeptide is or comprises an immunoglobulin G (IgG) heavy chain The process according to any one of the above.

  (6) Items 1 to 5 wherein the polypeptide encoded by the nucleic acid sequence is a human immunoglobulin G (IgG) heavy chain except for the amino acid residue at position +3 or −1 as defined in Item 1. The process according to any one of up to.

  (7) The process according to any one of items 1 to 6, further comprising the step of isolating and / or purifying the recombinant glycoprotein from the plant or the plant cell.

(8) A process for regulating glycosylation occupancy of an N-glycosylation site of a polypeptide after expression in a plant or plant cell, wherein the polypeptide comprises a consensus sequence Asn-X-Ser or Asn-X- Comprising the IgG heavy chain constant segment CH2 with said N-glycosylation site having Thr, X is any standard amino acid residue;
A nucleic acid sequence encoding the polypeptide, wherein a codon encoding a Tyr or Phe residue at position +3 counting from the Asn residue at position 0 of the N-glycosylation site is defined as Thr, Ser, Gly, Leu, Ile, Substituting a codon encoding an amino acid residue selected from Val and Met;
Expressing the nucleic acid sequence having the substitution made in the plant or plant cell in the previous step.

(9) A process for regulating glycosylation occupancy of an N-glycosylation site of a polypeptide after expression in a plant or plant cell, wherein the polypeptide comprises a consensus sequence Asn-X-Ser or Asn-X- Comprising the IgG heavy chain constant segment CH2 with said N-glycosylation site having Thr, X is any standard amino acid residue;
In the nucleic acid sequence encoding the polypeptide, a codon encoding the Tyr or Phe residue at position -1 as counted from the Asn residue at position 0 of the N-glycosylation site is the amino acid residue selected from Glu and Asp. Substituting with a codon encoding the group;
Expressing the nucleic acid sequence having the substitution made in the plant or plant cell in the previous step.

  (10) The plant is a higher plant, preferably a dicotyledonous plant, more preferably a Solanasae plant, more preferably a Nicotiana plant, and most preferably a Nicotiana benthamiana. Any one of items 1 to 9, wherein the plant cell is a cell derived from a higher plant, preferably a dicotyledonous plant, more preferably a solanaceous plant, more preferably a tobacco plant, most preferably a benthamiana tobacco. The process described in the section.

  (11) The item according to any one of items 1 to 10, further comprising the step of co-expressing a second nucleic acid sequence encoding a heterologous single subunit oligosaccharyltransferase in the plant, plant cell or plant cell. The process according to one paragraph.

  (12) The method according to item 11, wherein the oligosaccharide transferase of a heterologous single subunit is a Leishmania STT3A protein, STT3B protein, STT3C protein, STT3D protein, or a combination thereof.

  (13) The nucleic acid sequence encoding the polypeptide and the second nucleic acid sequence are both transiently expressed in the plant, preferably by transfection mediated by Agrobacterium, Item 11 Or the process according to 12.

  (14) using a first Agrobacterium containing a first DNA molecule comprising a T-DNA comprising a nucleic acid construct containing a DNA sequence of interest encoding a polypeptide having an N-glycosylation site; Co-transfecting said plant and a second DNA molecule comprising a T-DNA comprising a nucleic acid construct comprising a second DNA sequence encoding a heterologous single subunit oligosaccharyltransferase. Transfecting the plant with two Agrobacterium and expressing the first and second DNA sequences to produce a glycosylated form of the polypeptide as the glycoprotein. Item 11 wherein the nucleic acid sequence encoding the peptide and the second nucleic acid sequence are expressed in plants. The process according to any one of up to 13.

  (15) the glycoprotein comprises two different polypeptide chains (heterodimeric or heteromultimeric glycoprotein) and the process expresses the nucleic acid sequence of interest encoding the polypeptide of the first subunit Said polypeptide has an N-glycosylation site of the consensus sequence Asn-X-Ser or Asn-X-Thr, wherein X is any standard amino acid residue, wherein When the Asn residue of the N-glycosylation site is assigned to position 0 of the amino acid sequence, the amino acid residue at position +3 or position -1 may be as defined above, and a recombinant glycoprotein And expressing a nucleic acid sequence that encodes another polypeptide of another subunit of the process of any one of items 1-14.

  (16) A process for producing a recombinant protein in a plant, plant cell or plant cell, wherein a nucleic acid sequence encoding a polypeptide having an N-glycosylation site in the plant, plant cell or plant cell is produced. Expressing the nucleic acid sequence encoding a heterologous single subunit oligosaccharyltransferase.

  (17) A first DNA molecule comprising a T-DNA comprising a nucleic acid construct containing a DNA sequence of interest encoding a polypeptide having an N-glycosylation site, which is a process for producing a recombinant protein in a plant A nucleic acid construct containing a second DNA sequence encoding a heterologous single subunit oligosaccharyl transferase using a first Agrobacterium containing Transfecting the plant with a second Agrobacterium containing a second DNA molecule comprising DNA, and expressing the first and second DNA sequences as the glycoprotein Process as described above, which produces a glycosylated form of the polypeptide.

  (18) The process according to item 16 or 17, wherein the single subunit oligosaccharyltransferase is a Leishmania STT3A protein, STT3B protein, STT3C protein, STT3D protein or a combination thereof.

(19) A recombinant glycoprotein comprising a polypeptide comprising CH2 of an IgG heavy chain constant segment having a glycosylated N-glycosylation site of the consensus sequence Asn-X-Ser or Asn-X-Thr, Is any standard amino acid residue, where the Asn residue of the N-glycosylation site is assigned to position 0 of the amino acid sequence,
(A) the amino acid residue at position +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met, or (b) the amino acid residue at position -1 is selected from Glu and Asp ,
The above recombinant glycoprotein.

  (20) The recombinant glycoprotein according to item 19, wherein the recombinant glycoprotein is a glycoprotein expressed in a plant or plant cell.

  (21) The recombinant glycoprotein of item (a) has a glycosylation occupancy at the N-glycosylation site of at least 85%, more preferably at least 90%, and the glycoprotein of item (b) 21. Recombinant glycoprotein according to item 19 or 20, wherein has a glycosylation occupancy at said N-glycosylation site of at most 65%, preferably at most 55%.

  (22) The set according to any one of items 19 to 21, wherein the polypeptide includes an IgG heavy chain constant region, and preferably the polypeptide includes an IgG heavy chain including a variable region and a constant region. Changed glycoprotein.

  (23) The recombinant sugar according to any one of items 19 to 22, wherein the polypeptide is a human IgG heavy chain except for the amino acid residue at the +3 position or the -1 position defined in item 1. protein.

  (24) The recombinant glycoprotein according to item 22 or 23, wherein the polypeptide encodes any one human IgG heavy chain of G1, G2, G3 and G4 types.

(25) The polypeptide is
(I) the amino acid sequence of SEQ ID NO: 1 excluding the amino acid residues at positions +3 and −1 as defined in item 1;
(Ii) an amino acid sequence having a sequence identity with the amino acid sequence of SEQ ID NO: 1 of at least 95%, preferably at least 97%, and having amino acid residues at positions +3 and −1 as defined in item 1;
(Iii) There are substitutions, additions and / or deletions from 1 amino acid to 10 amino acids, preferably from 1 amino acid to 5 amino acids in the amino acid sequence of SEQ ID NO: 1. 25. A recombinant glycoprotein according to any one of items 19 to 24, comprising a heavy chain region comprising an amino acid sequence having amino acid residues.

  (26) The recombinant glycoprotein according to any one of items 19 to 25, which is an IgG antibody or an Fc fragment of an IgG antibody.

  (27) The recombinant glycoprotein according to item 19, which is an IgG antibody having a variable domain having affinity for CD20 or Her2 / Neu.

  (28) A pharmaceutical composition comprising the recombinant glycoprotein defined in any one of items 19 to 27 and a pharmaceutically acceptable carrier.

  (29) The recombinant glycoprotein according to any one of items 19 to 27 for use in treatment such as treatment of Her2 / Neu positive cancer.

  (30) A method for treating a patient suffering from Her2 / Neu-positive cancer, which comprises the step of administering to the patient an effective amount of the recombinant glycoprotein defined in item 27.

(31) A nucleic acid encoding a polypeptide comprising CH2 of an IgG heavy chain constant segment having the N-glycosylation site of the consensus sequence Asn-X-Ser, wherein X is any standard amino acid residue Here, when the Asn residue of the N-glycosylation site is assigned to position 0 of the amino acid sequence,
(A) the amino acid residue at position +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met, or (b) the amino acid residue at position -1 is selected from Glu and Asp ,
The nucleic acid.

  (32) The isolated nucleic acid of item 31, which encodes a human IgG heavy chain constant region except for the amino acid residues at positions +3 and −1 as defined in item 1.

  (33) A nucleic acid construct comprising a promoter active in plants and the nucleic acid according to item 31 or 32 operably linked thereto.

  (34) A vector comprising the nucleic acid construct of item 33.

  (35) A plant or plant cell comprising the nucleic acid construct of item 33 or the vector of item 34.

  (36) The plant or plant cell of item 35, further comprising a nucleic acid sequence encoding a heterologous single subunit oligosaccharyltransferase.

  (37) A plant or plant cell comprising a nucleic acid sequence encoding a heterologous single subunit oligosaccharyltransferase.

  (38) a first Agrobacterium containing a first DNA molecule comprising a T-DNA comprising a nucleic acid construct containing a DNA sequence of interest encoding a polypeptide having an N-glycosylation site, heterologous Agrobacterium comprising: a second Agrobacterium containing a second DNA molecule comprising a T-DNA comprising a nucleic acid construct comprising a second DNA sequence encoding a single subunit oligosaccharyltransferase Um mixture.

(39) A recombinant glycoprotein comprising a polypeptide that is an IgG heavy chain, said polypeptide having a glycosylated N-glycosylation site of the consensus sequence Asn-X-Ser or Asn-X-Thr Comprising the IgG heavy chain constant segment CH2, where X is any standard amino acid residue, where the Asn residue of the N-glycosylation site is assigned to position 0 of the amino acid sequence;
(A) the amino acid residue at position +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met, or (b) the amino acid residue at position -1 is selected from Glu and Asp ,
The above recombinant glycoprotein.

(40) A process for producing a recombinant glycoprotein in a plant or plant cell comprising the IgG heavy chain constant segment CH2 with the N-glycosylation site of the consensus sequence Asn-X-Ser or Asn-X-Thr Expressing a nucleic acid sequence encoding a polypeptide in said plant or plant cell, wherein X is any standard amino acid residue, wherein the Asn residue of said N-glycosylation site is an amino acid When assigned to the 0th position of the array,
(A) the amino acid residue at position +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met, or (b) the amino acid residue at position -1 is selected from Glu and Asp ,
The above process.

(41) A recombinant glycoprotein comprising a polypeptide comprising CH2 of an IgG heavy chain constant segment having a glycosylated N-glycosylation site of the consensus sequence Asn-X-Ser or Asn-X-Thr, X is any standard amino acid residue, where the Asn residue of the N-glycosylation site is assigned to position 0 of the amino acid sequence,
(A) the amino acid residue at position +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met, or (b) the amino acid residue at position -1 is selected from Glu and Asp ,
The above recombinant glycoprotein.

(42) A process for producing a recombinant glycoprotein in a plant, plant cell or plant cell, which encodes a polypeptide having an N-glycosylation site of the consensus sequence Asn-X-Ser or Asn-X-Thr Mutating the nucleic acid, wherein X is any standard amino acid residue, where the Asn residue of said N-glycosylation site is assigned to position 0 of the amino acid sequence and //-1 amino acid residue is the following item (a) or (b)
(A) the amino acid residue at position +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met, so that Thr is preferred, or (b) the amino acid residue at position -1 is Expressing the obtained (mutated) nucleic acid in the plant, the plant cell or the plant cell, thereby being selected from Glu and Asp, and thereby the recombination Producing the glycoprotein

  Mammalian proteins expressed in plants sometimes have a lower glycosylation occupancy at the N-glycosylation site than can be obtained by expression in animal expression systems. The inventors have surprisingly found that the Asn residue assigned to position 0 is position -1 as counted from the Asn residue of the N-glycosylation site of the conventional consensus sequence Asn-X-Ser / Thr. Alternatively, it has been found that glycan occupancy at the N-glycosylation site of proteins expressed in plants can be altered by appropriate selection of the amino acid residue at position +3. Preferably, the following amino acid residues: Thr, Ser, Gly, Leu, Ile, Val and Met are placed in the +3 position to increase or increase the N-glycosylation site as soon as it is expressed in plants. Glycosylation occupancy can be achieved. By placing the following amino acid residues: Glu and Asp at position −1, low or reduced glycosylation occupancy can be achieved at the N-glycosylation site after expression in plants. Thus, the present invention makes it possible to modify the glycosylation occupancy at the N-glycosylation site or protein as soon as it is expressed in plants. The present invention also makes it possible to produce recombinant glycoproteins having the desired glycosylation occupancy at the N-glycosylation site in plants or plant cells. The present invention also provides a recombinant glycoprotein having an amino acid substitution as indicated above at the N-glycosylation site of one or more consensus sequences Asn-X-Ser / Thr.

  We co-express glycosylation occupancy at the N-glycosylation site of glycoproteins expressed in plants or plant cells and heterologous single subunit oligosaccharyltransferases in the plants or plant cells. It has further been found that it can be increased by This embodiment can be employed in combination with the amino acid exchange at positions +3 and −1 as defined above or independent of it.

It is a sequence (SEQ ID NO: 1) of an immunoglobulin gamma 1 heavy chain constant region. The positions are numbered according to the EU index in Kabat [Kabat, E .; A. Et al., 1991, Sequences of Proteins of Immunological Interest (DHHS, Washington, DC), 5th edition]. N is the glycosylation site (shown in bold and underlined). The amino acid residue Asn at position 300 is the N-glycosylation site of the consensus sequence Asn-X-Ser / Thr, whose glycosylation occupancy is regulated in the present invention. The positions of amino acids used for amino acid substitutions made and described in the examples are shown in italics and bold. The number 300 shown is the amino acid sequence position +3 used in the present invention to increase glycosylation occupancy at the N-glycosylation site. The indicated numbered position 296 is amino acid sequence position -1 used in the present invention to reduce glycosylation occupancy at the N-glycosylation site. Amino acid sequence (SEQ ID NO: 2) of the cloned His tag version of the STT3-D gene (LmJF35.1160, accession number E9AET9) of Leishmania major. Added amino acid residues are underlined. FIG. 5 is a cloning scheme of nucleic acid modules encoding the amino acid sequences of rituximab and trastuzumab used to produce IgG heavy chains with constant regions with altered glycosylation. Type IIS restriction enzymes such as BsaI were used to seamlessly assemble nucleic acid sequence modules. Figure 2 is a cloning scheme of a corn codon optimized version of a large Leishmania STT3-D (LmJF35.1160, accession number E9AET9) with a C-terminal His tag. Type IIS restriction enzymes such as BsaI were used to seamlessly assemble nucleic acid sequence modules. SEQ ID NO: 21 is the amino acid sequence of STT3-D (LmJF35.1160, accession number E9AET9) of large Leishmania. SEQ ID NO: 22, which is the amino acid sequence of the cloned His-tag version of large Leishmania STT3-D. It is the schematic of the binary virus cloning vector [magnICON (trademark) system] based on plant virus TMV and PVX. The T-DNA defined by the left and right borders contains a plant active promoter (Act2 promoter), a viral RNA-dependent RNA polymerase coding sequence containing an intron, a viral movement protein coding sequence containing an intron, lacZα Contains a BsaI cloning site and a Nos terminator. Amino acid sequences of the variable region of recombinant trastuzumab (SEQ ID NO: 5) and the general light chain kappa constant region (SEQ ID NO: 6), and the nucleic acid sequences encoding them (SEQ ID NO: 3 and SEQ ID NO: 4, respectively). These are the amino acid sequences of the variable region (SEQ ID NO: 8) and heavy chain constant region (SEQ ID NO: 10) of recombinant trastuzumab IgG1, and the nucleic acid sequences encoding them (SEQ ID NO: 7 and SEQ ID NO: 9, respectively). Amino acid sequences of the variable region of recombinant rituximab (SEQ ID NO: 12) and the general light chain kappa constant region (SEQ ID NO: 14), and the nucleic acid sequences encoding them (SEQ ID NO: 11 and SEQ ID NO: 13, respectively). These are the amino acid sequences of the variable region (SEQ ID NO: 16) and heavy chain constant region (SEQ ID NO: 18) of recombinant rituximab IgG1, and the nucleic acid sequences encoding them (SEQ ID NO: 15 and SEQ ID NO: 17 respectively). It is an electrophoretic diagram of capillary gel electrophoresis (CGE) analysis under the reducing conditions of rituximab. HC is the heavy chain, LC is the light chain, and MabThera is the commercially available rituximab. The molar percentage of non-glycosylated and glycosylated variants of HC is shown at the top of each electropherogram. It is an electrophoretic diagram of capillary gel electrophoresis (CGE) analysis under the reducing conditions of plant-produced rituximab and its mutant variants. HC is the heavy chain, LC is the light chain, and Y300T is rituximab with an amino acid substitution at position 300 of the heavy chain corresponding to position +3 from the N-glycosyl site of the heavy chain constant region and Y being T. is there. Y300G is rituximab in which amino acid substitution at Y is performed at position 300 of the heavy chain. The approximate molar percentage of HC non-glycosylated and glycosylated variants is shown at the top of the corresponding peak of each electropherogram. It is an electrophoretic diagram of capillary gel electrophoresis (CGE) analysis under the reducing conditions of commercially available Herceptin, plant-produced trastuzumab and mutant variants thereof. HC is a heavy chain, LC is a light chain, and Y300T is trastuzumab in which amino acid substitution at Y is performed at position 300 of the heavy chain. The percentage of non-glycosylated and glycosylated variants of HC is shown at the top of the corresponding peak of each electropherogram. It is an electrophoretic diagram of capillary gel electrophoresis (CGE) analysis under the reducing conditions of commercially available Herceptin, plant-produced trastuzumab and mutant variants. HC is the heavy chain, LC is the light chain, and Y296E is trastuzumab amino acid substituted at position 296 of the heavy chain with Y being E. The percentage of non-glycosylated and glycosylated variants of HC is shown at the top of the corresponding peak of each electropherogram. FIG. 2 is an electrophoretogram of capillary gel electrophoresis (CGE) analysis under reducing conditions of MabThera (commercially available rituximab), plant-produced rituximab, and mutant variants thereof. HC is the heavy chain, LC is the light chain, and Y296E is rituximab with amino acid substitution at position 296 of the heavy chain, with Y being E. The percentage of non-glycosylated and glycosylated variants of HC is shown at the top of the corresponding peak of each electropherogram. It is an electrophoretic diagram of capillary gel electrophoresis (CGE) analysis of anti-CD20 mouse mIgG2a. HC is the heavy chain and LC is the light chain. It is an electrophoretic diagram of capillary gel electrophoresis (CGE) analysis of plant-produced rituximab and plant-produced trastuzumab when LmSTT3-D gene is coexpressed. HC is the heavy chain and LC is the light chain. The percentage of non-glycosylated and glycosylated variants of HC is shown at the top of the corresponding peak of each electropherogram. FIG. 4 is an alignment diagram of CH2 regions of various human IgG subclasses. The sequence segment containing the N-glycosylation site is boxed. The N-glycosylation site Asn-X-Ser / Thr is indicated by a gray background. The amino acid residue positions are numbered according to the EU index in Kabat (Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, DHHS, Washington, DC, 5th edition). The first column of IgHG1 ^* 01 is SEQ ID NO: 19. The last column shows the CH2 region of allele A of the mouse IgG2 subclass. FIG. 4 is an alignment diagram of heavy chain constant regions of various IgG subclasses. The first column of IgHG ^* 01 is SEQ ID NO: 20. FIG. 4 is an electrophoretic diagram of capillary gel electrophoresis (CGE) analysis under reducing conditions of plant-produced rituximab and plant-produced trastuzumab with and without co-expression of large Leishmania oligosaccharyltransferase LmSTT3-D. . HC is the heavy chain and LC is the light chain. The percentage of non-glycosylated and glycosylated variants of HC is shown at the top of the corresponding peak of each electropherogram. Electrophoretic diagram of capillary gel electrophoresis (CGE) analysis under reduced conditions of plant-produced palivizumab and plant-produced anti-Ebola mAb with and without co-expression of large Leishmania oligosaccharyltransferase LmSTT3-D It is. HC is the heavy chain and LC is the light chain. The percentage of non-glycosylated and glycosylated variants of HC is shown at the top of the corresponding peak of each electropherogram.

  The process of the invention makes it possible to produce recombinant glycoproteins by expression in plants or plant cells. However, the recombinant glycoprotein of the present invention is not limited to expression in plants or plant cells. In general, recombinant sugars based on various production hosts (bacteria, fungi, animals, insects and plant cells) and expression vectors designed for either stable transgenic or transient expression. Many different expression systems can be used to produce the protein. Such systems are generally known to those skilled in the art and have been described in the literature [for review see below: Huang, C .; J. et al. Lin, H .; And Yang, X .; 2012, J. Org. Ind. Microbiol. Biotechnol. 39: 383-399; Hou, J .; Tyo, K .; E. Liu, Z .; 2012, FEMS Yeast Res. 12: 491-510; Martinez, J .; L. Liu, L .; Petranovic, D .; Et al., 2012, Curr. Opin. Biotechnol. April 12, (electronic publication prior to printing); Su, X. Schmitz, G .; Zhang, M .; 2012, Adv Appl Microbiol. 81: 1-61; Ghaderi, D .; Zhang, M .; Hurtado-Ziola, N .; And Varki, A .; 2012, Biotechnol. Genet. Eng. Rev. 28: 147-175; Egelkrout, E .; Rajan, V .; And Howard, J. et al. A. 2012, Plant Sci. 184: 83-101]. The choice of expression system depends on various factors such as the cost of the material to produce the protein and the desired speed and scale.

  In one embodiment, the recombinant glycoprotein is produced in a plant or plant cell according to the production or modification process of the present invention. Among plant expression systems, transient expression systems based on plant viruses are speed or production, yield and versatility for the production of various types of recombinant proteins, including heteromultimeric proteins such as monoclonal antibodies. This is advantageous. Another advantage of the plant expression system is that it can confer the production of plant virus particles by expressing the plant virus coat protein or fusion protein from the expression vector (Werner, S. et al., 2006, Proc. Natl. Acad.Sci.USA, 103: 17678-17683; WO2007 / 031339). Plant expression systems suitable for the present invention have been described in numerous research papers, reviews and patent documents (eg, Marilonnet, S., Theeringer, C., Kandzia, R. et al., 2005, Nat. Biotechnol., 23: 718-723; Giritch, A., Marylonnet, S., Engller, C. et al., 2006, Proc. Natl. Acad. Sci. USA, 103: 14701-14706; Gleba, Y., Klimyuk, V. and Marilonnet, S., 2007, Curr. Opin.Biotechnol., 18: 134-141; Klimyuk, V., Pogue, G., Herz, S. et al., 2012, Curr Top Microbiol Immunol., 4 15 days; WO2005 / 049839; WO2006 / 079546). In WO2005 / 049839, detailed information of a possible plant virus expression vector is listed including its sequence information. Viral vector design, cloning strategy and expression of recombinant proteins including immunoglobulins are described in detail in WO002006 / 079546 and in Example 1 herein.

  Cloning of nucleic acid sequences encoding polypeptides to be expressed is part of the general knowledge of those skilled in the art. A particularly convenient cloning strategy is modular cloning to seamlessly bind different DNA fragments and this cloning has been established in our laboratory (Engler, C., Kandzia, R. and Marylonnet, S., 2008, PLoS One, 3: e3647; Weber E., Engler, C., Guetzner, R. et al., 2011, PLoS One, 6: e16765; Methods Mol Biol., 729: 167-81; Thiem, F., Engler, C., Kandzia, R., et al. This system is simple, reliable, convenient to use, and allows any complex construct to be manipulated quickly. The overall scheme for seamlessly assembling a binary vector in T-DNA containing a nucleic acid sequence encoding the IgG heavy chain used for rituximab and trastuzumab from four gene modules and one viral vector is shown in FIG. 2A. The seamless assembly of heavy chain modules is achieved by type IIS restriction enzymes. In this case, BsaI was used. The polypeptide to be expressed can be provided by placing a coding sequence encoding a signal peptide at the 5 'end of the nucleic acid sequence encoding the polypeptide using an N-terminal signal peptide. In the following examples, this strategy was used to assemble the IgG heavy chain of interest and various Fc variant variants and clone them into appropriate viral vectors. To express the antibody, the nucleic acid sequence encoding the heavy chain (HC) may be cloned into a TMV based vector and the nucleic acid sequence encoding the light chain (LC) may be cloned into a PVX based vector ( (See FIG. 3). In the following examples, a binary vector that can be transformed into plant cells using Agrobacterium was used. Other transformation methods can also be used. The final expression vector may be tested for expression in plants.

  Expression of a nucleic acid sequence (polynucleotide) encoding a polypeptide of the invention in a plant or plant cell is generally accomplished using a nucleic acid molecule (also referred to herein as a vector) comprising a nucleic acid construct containing the nucleic acid sequence, Transforming the plant or plant cell. A nucleic acid sequence encoding a polypeptide having an N-glycosylation site that is glycosylated is also referred to herein as a “nucleic acid sequence of interest”. When expressing two or more different polypeptides each having an N-glycosylation site, for example when expressing to produce a heterodimeric or heteromultimeric glycoprotein, two or more of interest There are different nucleic acid sequences.

  A nucleic acid molecule is generally a DNA molecule. In this case, the target nucleic acid sequence contained therein is DNA. An example of a DNA molecule is a binary vector that can be transformed into plant cells by Agrobacterium-mediated transformation. Another example of a nucleic acid molecule is a DNA vector that is transformed by particle bombardment.

  Recombinant glycoprotein polypeptides may be expressed during the process of the invention in stably transformed transgenic plants or cells thereof. “Stablely transformed” in the case of plant cells means that the nucleic acid sequence (s) of interest is incorporated into the chromosomal DNA so that it is transferred to daughter cells. “Stablely transformed” in the case of (all) plants means the nucleic acid sequence or sequences of interest in which all somatic cells of the plant are incorporated into the chromosomal DNA so that they can be passed on to the progeny plants. Is contained. Preferably, however, the recombinant glycoprotein is expressed by transient expression from a transiently transformed plant. The term “transient” uses a nucleic acid molecule (vector) or nucleic acid construct, for example, using a selectable agent and a selectable marker gene that can detoxify the selectable agent. Means that no selection method is used to select transfected cells or plants from non-transfected cells or plants. As a result, transfected nucleic acids are generally not stably introduced into plant chromosomal DNA. Transient expression methods take advantage of the effects of transfection on just transfected plant cells. Transient expression is suitable for rapid expression, among others, as described above. Furthermore, since there is no need for a selectable marker gene for selecting plant cells or plants, and therefore there is no need to insert antibiotic resistance genes or herbicide resistance genes into the plants, In the environment where they are placed, such gene spread can be avoided. In the case of transient transfection of plants by Agrobacterium-mediated transfection, the T-DNA of the binary vector or Ti-plasmid preferably has no selectable marker gene, so that such a marker gene is It is not inserted into plant cells.

  Various methods are known for introducing nucleic acid molecules such as DNA molecules into plants or plant cells for transient expression. In the present invention, Agrobacterium is preferably used for transfecting plants with nucleic acid molecules (vectors) or nucleic acid constructs, for example by agroinfiltration. A system and method for large-scale infiltration of plants using Agrobacterium is described in WO2009 / 095183. In another embodiment, a plant or plant part is sprayed with a suspension containing cells of an Agrobacterium strain, but this method can be applied on a large scale to a large number of plants, such as field plants. Very suitable for. Such a process of spray transfection is described inter alia in WO2012 / 019660.

  Agrobacterium strains are commonly used for plant transformation and transfection and are known to those skilled in the art from general knowledge, Agrobacterium tumefaciens or Agrobacterium rhizogenes Belonging to the species. The Agrobacterium strain used in the process of the present invention contains a DNA molecule (Ti plasmid) as the nucleic acid molecule, and the nucleic acid molecule contains a nucleic acid construct containing the DNA sequence of interest. The DNA sequence of interest may encode a polypeptide of the invention or one or more expressed polypeptides. The nucleic acid construct is typically present in the T-DNA of the Ti plasmid in order to introduce the nucleic acid construct into the plant cell by the secretion system of the Agrobacterium strain. On at least one side or on both sides, the nucleic acid construct is adjacent to the T-DNA border sequence, allowing transfection of the plant (s) and introduction of the nucleic acid construct into cells of the plant. . Preferably, the nucleic acid construct is present in T-DNA and is flanked by T-DNA border sequences on both sides.

  Preferably, the nucleic acid construct is present in the T-DNA of the Ti plasmid of the Agrobacterium strain. The Ti plasmid may contain a selection marker outside the T-DNA in order to enable bacterial cloning and genetic manipulation. However, if present, the T-DNA transferred into the plant cell preferably does not include a selection marker that allows selection of the plant or plant cell containing the T-DNA. In this embodiment, examples of selection marker genes that should not be present in the T-DNA of a DNA molecule (Ti plasmid) are antibiotic resistance genes or herbicide resistance genes. Where the process of the invention uses transient transfection and expression of the nucleic acid sequence of interest (or of the polypeptide), the process uses such an antibiotic resistance gene or herbicide resistance gene Does not include the step of selecting plant cells incorporating the nucleic acid molecules of the invention. Therefore, it is not necessary to incorporate antibiotic resistance genes or herbicide resistance genes into the plant, so that in the process of the present invention it is unlikely that such genes will spread to the environment.

  As indicated above, in order to express a heteromultimeric protein such as an antibody as the glycoprotein of the present invention, all subunits (polypeptide chains) are preferably present in the same cell of a plant or plant cell. It may be expressed over time. This is done by co-transfecting plants or plant cells with a mixture of Agrobacterium for each subunit or polypeptide. Therefore, the present invention provides a process for producing in a plant a recombinant glycoprotein comprising two or more (preferably two) different polypeptide chains, such as an IgG antibody, Transfecting a plant with Agrobacterium containing a DNA molecule comprising a T-DNA comprising a nucleic acid construct containing a DNA sequence of interest encoding a polypeptide of one subunit comprising the steps of: The polypeptide has an N-glycosylation site (as defined above, with amino acid residues at positions +3 and -1 as defined herein) and the glycoprotein separation An (another) DNA molecule comprising an (another) T-DNA comprising a nucleic acid construct containing an (another) DNA sequence encoding a polypeptide of a subunit of Using Agrobacterium, comprising the steps of transfecting said plant, a step of expressing the DNA sequence to produce glycoproteins. The polypeptide of another subunit may or may not contain an N-glycosylation site. If the polypeptide has an N-glycosylation site, it may or may not have the amino acid residues at positions +3 and -1 as defined herein. Preferably, the polypeptide of another subunit having an N-glycosylation site is also a +3 position as defined herein and in order to achieve high N-glycan occupancy at all N-glycosylation sites. It has an amino acid residue at position -1.

  However, it is also possible to express multiple polypeptides of multiple subunits of heteromultimeric proteins separately in different plants or plant cells. Separately expressed polypeptides, at least one of which is produced according to the process of the present invention, may be separately purified and reconstituted in vitro to form a recombinant heteromultimeric protein.

  The nucleic acid construct contains the nucleic acid sequence of interest so that it can be expressed in plant cells. For this reason, in the nucleic acid construct, the target nucleic acid sequence may be under the control of a promoter active in plant cells. Examples of nucleic acid sequences of interest are DNA virus replicons or RNA virus replicons, or DNA encoding the gene to be expressed. Genes produce polypeptides when expressed in plants. A viral replicon may also encode a polypeptide that is expressed in the plant (s) cells. In addition to the nucleic acid sequence, the nucleic acid construct includes other sequences such as a regulatory sequence for expressing the nucleic acid sequence of interest, such as a transcriptional promoter and terminator. The nucleic acid sequence may additionally encode a signal peptide that is expressed as the N-terminal signal peptide of the polypeptide. The nucleic acid construct may further contain a gene to be expressed, such as a gene encoding a gene silencing suppressor such as P19 protein. The expression of such further included genes can be under the control of the same or different promoter as the promoter used to express the protein of the invention. Agrobacterium-mediated gene transfer and vectors therefor are known to those skilled in the art, for example, references cited above, or Slater, Scott and Fowler, Plant Biotechnology, 2nd edition, Oxford University Press, 2008. Due to plant biotechnology textbooks.

  As used herein, a “promoter active in a plant cell” means a DNA sequence capable of controlling (initiating) transcription in the plant cell. This includes any promoter of plant origin, but also includes promoters of non-plant origin capable of inducing transcription in plant cells, ie certain promoters from viruses or bacteria, eg cauliflower mosaic Virus 35S promoter (CaMV35S promoter) [Harpster et al. (1988) Mol Gen Genet. , 212 (1): 182-90], but not limited to, but not limited to, the seed-specific promoter (eg WO89 / 03887) , Organ primordium specific promoter [An et al. (1996) Plant Cell 8 (1): 15-30], stem specific promoter [Keller et al. (1988) EMBO J. et al. 7 (12): 3625-3633], leaf-specific promoters [Hudspeth et al. (1989) Plant Mol Biol. 12: 579-589], mesophyll-specific promoters (such as the light-inducible Rubisco promoter), root-specific promoters [Keller et al. (1989) Genes Dev. 3: 1639-1646], tuber specific promoter [Keil et al. (1989) EMBO J. Biol. 8 (5): 1323-1330], vascular tissue specific promoter [Peleman et al. (1989) Gene 84: 359-369], male selection promoter (WO89 / 10396, WO 92/13956), cleavage site specific And tissue-specific or organ-specific promoters, such as typical promoters (WO 97/13865). For transient expression, a constitutive promoter is preferably used. However, the constitutive promoter may be tissue specific or organ specific, and in one embodiment is not tissue specific or organ specific. Suitable promoters are those used in the examples described hereinafter.

  Protein glycosylation occurs in the eukaryotic ER and / or Golgi apparatus. In order to direct the newly created polypeptide to these compartments, a polypeptide with an N-glycosylation site to be glycosylated should be equipped with an appropriate signal peptide at the N-terminus. The use of such signal peptides to direct the ER and / or Golgi is known. Appropriate signal peptides are used to express beta (1,4) -galactosyltransferase in plants, and such expression is described in several publications cited hereinafter.

  As used herein, the term “construct” refers to a recombinant construct comprising a nucleotide sequence of interest. Preferably, the construct encodes a polypeptide having at least an N-glycosylation site comprising amino acid residues at positions +3 and -1 according to the present invention. In addition, other nucleic acid sequences encoding the polypeptide may also be included in the same or different constructs.

  In embodiments where it is desirable to strongly express the polypeptide, the nucleic acid construct may encode a viral vector that replicates in plant cells to form a viral vector replicon. To be replicated, viral vectors and replicons contain an origin of replication that can be recognized by nucleic acid polymerases present in plant cells, such as viral polymerases expressed from the replicon. The viral vector may be an RNA viral vector because the RNA viral vector forms an RNA replicon. In the case of an RNA viral vector, a replicon may be formed from a DNA construct after introduction into the nucleus of the plant cell by transcription under the control of a promoter active in the plant cell. In the case of a DNA replicon, the DNA replicon is formed by recombination between two recombination sites adjacent to the sequence encoding the viral replicon of the DNA construct, eg as described in WO00 / 17365 and WO99 / 22003. May be. If the replicon is encoded by a DNA construct, an RNA replicon is preferred. The use of DNA and RNA viral vectors (DNA or RNA replicons) has been extensively described in the literature for many years. Some examples are the following patent publications: WO 2008/028661, WO 2007/137788, WO 2006/003018, WO 2005/071090, WO 2005/049839, WO 02/0970880, WO 02/0888369, WO 02/068664. An example of a DNA viral vector is based on geminivirus. Viral vectors or replicons based on plant RNA viruses, in particular those based on positive-sense single-stranded RNA viruses, can be used in the present invention. Thus, the viral replicon may be a positive sense single stranded RNA replicon. Examples of such viral vectors are those based on tobacco mosaic virus (TMV) and potex virus X (PVX). “Based on” means that the viral vector uses a replication system for these viruses. Virus vectors and expression systems based on potexviruses are described in EP2061890 or WO2008 / 028661. A number of other plant virus replicons are described in the aforementioned patent publications.

  Agrobacterium strains that can be used in the present invention are those commonly used in the art to transfect or transform plants. In general, binary vector systems and binary strains are used. That is, the vir gene necessary for transferring T-DNA to plant cells on one side and the T-DNA on the other side are mounted on separate plasmids. Examples of Agrobacterium strains that can be used are provided in the paper by Hellens et al., Trends in Plant Science 5 (2000) 446-451 for binary Agrobacterium strains and vector systems. In the context of binary Agrobacterium strains, the plasmid containing the vir gene is referred to as the “vir plasmid” or “vir helper plasmid”. The plasmid containing the T-DNA to be transfected is a so-called binary vector, also referred to herein as a “DNA molecule” or “vector”. The term “strain” or “Agrobacterium strain” refers to an Agrobacterium component other than a binary vector. Therefore, in this specification, a binary Agrobacterium strain that does not contain a binary vector and a product after the introduction of the binary vector are referred to by the same strain name.

  Plants can be transiently transfected by subjecting a part of the plant to a suspension, preferably an aqueous suspension, containing cells of an Agrobacterium strain containing the nucleic acid molecules described above. . A suspension of Agrobacterium can be produced as follows. A DNA molecule or vector containing the nucleic acid construct may be transformed into an Agrobacterium strain, and the transformed Agrobacterium culture is optionally at a selective pressure to maintain the DNA molecule. It may be grown under application. In one method, the Agrobacterium strain used is then seeded in culture medium and allowed to grow to a high cell density. In order to obtain a large amount of culture medium, a small-scale high-density culture medium may be seeded on a larger scale culture. In general, Agrobacterium is grown to a cell density corresponding to an OD of at least 1 at 600 nm, typically about 1.5. Such a dense Agrobacterium suspension is then diluted to reach the desired cell density used for transfection. Water is used to dilute the dense Agrobacterium suspension. The water can include a buffer or salt. The water may further comprise a surfactant as described in WO2012 / 019660.

Preferred embodiments of the production process of the present invention using transient expression based on Agrobacterium are inter alia the following two processes:
A process for producing a recombinant glycoprotein in a plant, using a suspension containing cells of an Agrobacterium strain containing a nucleic acid molecule comprising a nucleic acid construct containing a nucleic acid sequence, to transfect the plant or a part thereof. The nucleic acid sequence encodes a polypeptide, wherein the polypeptide has an N-glycosylation site of the consensus sequence Asn-X-Ser / Thr, where X is any standard amino acid A residue, wherein the Asn residue of the N-glycosylation site is assigned to position 0 of the amino acid sequence,
(A) the amino acid residue at position +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met, or (b) the amino acid residue at position -1 is selected from Glu and Asp.
The above process,
A process for producing a recombinant glycoprotein in a plant, wherein an aqueous suspension containing cells of an Agrobacterium strain comprising a nucleic acid molecule comprising a nucleic acid construct containing a nucleic acid sequence is applied to a part of said plant Wherein the nucleic acid sequence encodes a polypeptide comprising CH2 of an IgG heavy chain constant segment having an N-glycosylation site of the consensus sequence Asn-X-Ser / Thr, where X is any standard An amino acid residue, wherein the Asn residue of the N-glycosylation site is assigned to position 0 of the amino acid sequence,
(A) the amino acid residue at position +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met, or (b) the amino acid residue at position -1 is selected from Glu and Asp ,
The above process,
A process for producing a recombinant glycoprotein, such as an IgG antibody, comprising two or more different polypeptide chains (subunits) in a plant, encoding a polypeptide of the first subunit of the glycoprotein, of interest Transfecting the plant with Agrobacterium containing a DNA molecule comprising T-DNA comprising a nucleic acid construct containing a DNA sequence, wherein the polypeptide comprises a consensus sequence Asn-X-Ser or Has an Asn-X-Thr N-glycosylation site, where X is any standard amino acid residue, wherein the Asn residue of said N-glycosylation site is assigned to position 0 of the amino acid sequence In the case where the amino acid residue at position +3 or −1 is as defined above and another sub-unit of the glycoprotein. Transfecting said plant with Agrobacterium containing a DNA molecule comprising a T-DNA comprising a nucleic acid construct comprising a DNA sequence encoding a polypeptide of: and expressing the glycoprotein with a glycoprotein Producing the process.

  The polypeptide expressed in the process of the present invention is generally heterologous to the plant expressed in (or in its cells) within the present invention. That is, the plant or part thereof does not naturally produce the protein. It is possible to optimize the codon usage of the nucleic acid sequence of interest encoding the polypeptide (s) for expression in plants, in particular the plants employed for expression of the polypeptide, desirable. Codon optimization is a standard method for increasing the expression yield of proteins such as animal proteins in plants. Codon-optimized genes and nucleic acid sequences can also be ordered from commercial sources such as Enterochon GmbH, Regensburg, Germany for expression in plants or plants of a particular genus or species.

  Plants or cells thereof that can be used for polypeptide expression and recombinant glycoprotein production are not particularly limited. Suitable are multicellular plants, especially higher plants. Either monocotyledonous or dicotyledonous can be used. Plants that are not used for the production of human food or animal food are preferred. For example, tobacco species such as tobacco (Nicotiana tabacum) and Bensamiana tobacco. The latter is most preferred.

  There are no particular limitations on the present invention for glycans added to the N-glycosylation site of a polypeptide. As described in more detail below, the type of glycan produced and added will depend on the organism used to express the polypeptide. Also among plants, the glycans added depend on whether the plant has a native glycosylation system or a genetically engineered glycosylation system. Thus, as used herein, an N-glycosylation site of a polypeptide is either N-glycosylated or N-linked glycan when the N-glycosylation site carries any glycan of any size. ("N-glycan").

  Plants naturally produce glycoproteins, including glycoproteins with N-linked glycans. Therefore, plants naturally have the mechanisms necessary for this type of post-translational modification. N-glycosylation of a polypeptide that is expressed according to the invention to form a recombinant glycoprotein therefore occurs in plant cells that express the polypeptide. The plant or cells thereof used in the process of the present invention may have a wild-type plant glycosylation mechanism. In this case, the N-glycan added to the N-glycosylation site becomes a plant-type glycan. Plant-type glycans differ in some respects from those produced in animal cells. In plants, beta (1,2) -xylose and alpha (1,3) -fucose residues have been shown to be linked to the glycan core Man3GlucNAc2-Asn, while they are mammalian. Instead of being detected in glycans, sialic acid residues and terminal beta (1,4) -galactosyl structures are generated instead. The unique N-glycans added to the plant affect both the immunogenicity and functional activity of the protein and may therefore imply limitations on using the plant as a protein production platform. Indeed, the immunogenicity of beta (1,2) -xylose residues and alpha (1,3) -fucose in mammals has been described (Bardor et al., 2003, Glycobiology 13: 427). The enzyme that catalyzes the transfer of xylose from UDP-xylose to the core β-linked mannose of N-glycans bound to proteins is beta (1,2) -xylosyltransferase (“XyIT”, EC 2.4. 2.38). Beta-1,2-xylosyltransferase is an enzyme that is unique to plants and some invertebrate species and does not occur in humans or other vertebrates. WO 2007/107296 describes the identification and cloning of beta-1,2-xylosyltransferases from the genus Tobacco such as Bensamiana tobacco. The enzyme that catalyzes the transfer of fucose from GDP-fucose to the core β-linked N-acetylglucosamine (GlcNAc) of the N-glycan bound to the protein is alpha (1,3) -fucosyltransferase (“FucT”). EC 2.4.1.214). WO2009 / 056155 describes the cDNA sequence of alpha (1,3) -fucosyltransferase from Bensamiana tobacco. Therefore, in order to avoid N-glycans containing plant-type beta (1,2) -xylose residues and / or alpha (1,3) -fucose in N-glycosylation, beta-1,2-xylosyl Plants (or their cells) that do not express transferase and / or alpha (1,3) -fucosyltransferase may each be employed in the present invention. Such plants and their use have been described. WO 2008/141806 describes knockout bodies with two alpha (1,3) -fucosyltransferase genes and one beta (1,2) -xylosyltransferase gene of Arabidopsis thaliana. WO 2009/056155 describes an RNA interference strategy for generating benthamiana tobacco plants that lack the formation of beta-1,2-xylose structures on heterologous glycoproteins and are also free of alpha-1,3-fucose structures. Has been. Yin et al. (2011, Protein Cell 2:41) reported in tobacco that the RNA interference (RNAi) strategy was used to down-regulate endogenous xylosyltransferase and fucosyltransferase expression. They found that glycosylated and core fucosylated N-glycans in the glycoengineered series are greatly but not completely reduced. WO2010 / 145846 describes knockout bodies at two beta (1,2) -xylosyltransferase genes of N. benthamiana tobacco. Homozygous combination of four alleles without beta (1,2) -xylosyltransferase is sufficient to completely eliminate beta-1,2-xylosyltransferase activity in N. benthamiana tobacco It was proved. WO 2013/050155 describes a plant of the genus Tobacco that lacks the activity of alpha (1,3) -fucosyltransferase and beta (1,2) -xylosyltransferase, which plant is a heterologous sugar according to the present invention. It can be applied as a host plant or host cell for producing a protein.

  Alternatively or preferably, additionally, the plant used in the process of the invention may be derived from an animal or human in order to add a beta (1,4) -galactosyl structure. It may be genetically engineered to express galactosyltransferase. Beta (1,4) -galactose is derived from human beta (1,4) -galactosyltransferase I (GalT) (Bakker et al., 2001, Proc. Natl. Acad. Sci. USA 98: 2899), and chicken and zebrafish beta ( It has been introduced into plant-produced glycoproteins by expressing 1,4) -galactosyltransferase I (WO2008 / 125972). Bakker et al. (2006, Proc. Natl. Acad. Sci. USA 103: 7577) and WO 2003/078637 include human GalT and the cytoplasmic tail, transmembrane domain and stem region (CTS domain) of Arabidopsis xylosyltransferase (XylT). ) And fusions are described. They also found that in tobacco, the addition of the CTS domain caused a significant decrease in N-glycans with xylose and fucose residues attached to the core. In Vezina et al. (2009, Plant. Biotechnol. J. 7: 442) and WO 2008/151440, N-acetylglucosaminyltransferase I (GNTI) from tobacco is used to adapt GalT activity to the early secretory pathway of plants. GalT was fused to the membrane tethering domain. Glycans obtained from a Bensamiana tobacco plant expressing the GNTI-GalT fusion consist of galactosylated and non-galactosylated hybrid and immature oligomannose N-glycans, which can be detected alpha (1 , 3) -fucose residues and beta (1,2) -xylose residues were not contained. In WO2008 / 125972, the chicken and zebrafish CTS domains were replaced with the rat sialyltransferase CTS. Zebrafish GalT substituted at the amino terminus with the rat sialyltransferase CTS region produced mainly biantennary, doubly galactosylated N-glycans in Bensamiana tobacco. In Strasser et al. (2009, J. Biol. Chem. 284: 20479), human GalT was fused to the rat sialyltransferase CTS domain. This fusion protein lacks plant-specific beta (1,2) -xylosyltransferase and core alpha (1,3) -fucosyltransferase activities and expresses an anti-human immunodeficiency virus antibody. It was expressed in Samiana tobacco. EP2551348A describes the production of galactosylated N-glycans in plants. EP 1137789 A describes the expression of beta (1,4) -galactosyltransferase in plants.

  In a preferred embodiment, a plant that expresses beta (1,4) -galactosyltransferase and has no or little activity of alpha (1,3) -fucosyltransferase and beta (1,2) -xylosyltransferase (or The cells) are used.

  Glycoproteins can be purified using generally known methods after being produced in plants or plant cells. In one embodiment, the glycoprotein is an immunoglobulin IgG that can be purified using protein A affinity chromatography, more preferably IgG1. Purification can then include other types of column chromatography, if necessary, using a matrix that has an affinity for the purification tag. A detailed description of the purification protocol used for two representative IgG1 subclass immunoglobulins, rituximab and trastuzumab is given in Examples 2 and 3, respectively. Purification methods and detailed protocols for immunoglobulins belonging to various classes and subclasses, particularly IgG classes, are known in the art of academia and industry and have been described in numerous publications [Danielsson, A. et al. Et al., 1988, J. Am. Immunol. Meth. 115: 79-88; Kleine-Tube, J .; Et al., 1995, J. MoI. Immunol. Meth. 179: 153-164; Denizli, A .; Et al., 1995, J. MoI. Chrom. 668: 13-19; Huse, K .; Et al., 2002, J. MoI. Biochem. Biophys. Meth. 51: 217-231; McAthee, C .; P. And Hornbuckle, J. et al. 2012, Curr. Prot. Protein Sci. Chapter 8: Unit 8.10; for an overview, Marichal-Gallardo, P .; A. And Alvarez, M .; M.M. 2012, Biotechnol. Prog. 28: 899-916]. In addition, kits for purifying Ig are commercially available from many companies (for example, GE HealthCare, Promega, Thermo Scientific, etc.). A book on antibody purification is "Process Scale Purification of Antibodies", edited by Uwe Gottschalk, John Wiley & Sons, Hoboken, NJ, 2009.

  In the present invention, N-glycosylation site N-glycosylation site of the recombinant glycoprotein consensus sequence Asn-X-Ser or Asn-X-Thr (also abbreviated herein as “Asn-X-Ser / Thr”). Glycan occupancy is expressed in plants or plant cells by placing a specific amino acid residue at position +3 or −1 from the N-glycosylation site, where X is a standard 20 amino acid residue It can be adapted as soon as it is done. The term “N-glycosylation site” is present in the well-known consensus sequence “Asn-X-Ser / Thr” of N-linked glycosylation of proteins or polypeptides as soon as expressed in eukaryotic cells. Asparagine residue. “Position +3” means the position of the third amino acid residue of the polypeptide from the N-glycosylation site toward the C-terminus, whereby the Asn residue of the N-glycosylation site is assigned position 0. It is done. "Position -1" means the position of the first amino acid residue of the polypeptide in the N-terminal direction from the N-glycosylation site, whereby the Asn residue of the N-glycosylation site Assigned.

  “Glycan occupancy” or “glycosylation occupancy” of a selected N-glycosylation site refers to a fraction of recombinant glycoprotein molecules in a composition comprising a plurality of recombinant glycoprotein molecules, wherein And the N-glycosylation site carries any glycans. The type of glycan structure linked to the N-glycosylation site is not limited. If a given polypeptide has multiple N-glycosylation sites in its amino acid sequence, the occupancy is defined separately for each of these multiple N-glycosylation sites.

  To achieve high or increased N-glycan occupancy at a given N-glycosylation site, the amino acid residue at position +3 is any one of Thr, Ser, Gly, Leu, Ile, Val and Met. One. Thr, Ser and Gly are preferred among these residues, Thr and Ser are more preferred, and Thr is most preferred. However, the residue at position +3 may be any of these residues.

  To achieve low or reduced N-glycan occupancy at a given N-glycosylation site, the amino acid residue at position -1 is either one of Glu and Asp. However, it is preferred to provide for high or increased N-glycan occupancy.

  Increased N-glycan occupancy is a glycoprotein expressed in plants or plant cells that has another amino acid residue at position +3 when the amino acid residue at position +3 is as defined above. And otherwise it means a higher N-glycan occupancy compared to glycoproteins expressed under the same conditions. Decreased N-glycan occupancy is a glycoprotein expressed in plants or plant cells, where the amino acid residue at position -1 is as previously defined and has another amino acid residue at that position. And otherwise, it means a lower N-glycan occupancy compared to polypeptides expressed under the same conditions.

  N-glycan occupancy may be determined experimentally, for example using capillary gel electrophoresis as performed in the examples. A qualitative analysis of increased or decreased N-glycan occupancy is often sufficient. The recombinant glycoprotein of item (a) may have a glycosylation occupancy at the N-glycosylation site of at least 85%, more preferably at least 90%, more preferably at least 95%. The recombinant glycoprotein of item (b) may have a glycosylation occupancy at the N-glycosylation site of at most 65%, preferably at most 55%.

  If the polypeptide has multiple N-glycosylation sites, all or some may be manipulated to have high or low N-glycan occupancy or may be manipulated separately.

  The present invention also provides a process for producing a recombinant glycoprotein in a plant, plant cell or plant cell, wherein the process obtains the desired amino acid residue at position +3 and / or -1. For genetic manipulation. The process provides a nucleic acid encoding a polypeptide having an N-glycosylation site of the consensus sequence Asn-X-Ser or Asn-X-Thr, wherein X is any standard amino acid residue And when the Asn residue of the N-glycosylation site is assigned to position 0 of the amino acid sequence, the amino acid residues at positions +3 and / or −1 are defined in the present specification ( a step of manipulating a nucleic acid as defined in a) or (b) and expressing the engineered nucleic acid in a plant, plant cell or plant cell thereby recombining Producing a glycoprotein.

  The recombinant glycoprotein may be a monomeric glycoprotein or a multimeric glycoprotein. Recombinant glycoprotein formation includes expression of at least one polypeptide of the invention in plants, plant cells or plant cells, N-glycosylation, folding, optionally further post-translational modifications, or Including processes such as directing to specific cell compartments or apoplasts. However, this does not exclude the possibility of obtaining the recombinant glycoprotein according to the invention by other means or methods. When the recombinant glycoprotein is a multimer, it may be a homomultimer or a heteromultimer. When it is a heteromultimeric recombinant glycoprotein, only one or more subunits may have an N-glycosylation site and may be N-glycosylated. Thus, only one of the subunits may have a modified N-glycosylation site as defined herein. Multiple subunits of a heteromultimeric protein may be co-expressed in plants or plant cells, for example as described in WO002006 / 079546. Suitable heteromultimeric proteins are immunoglobulins, especially immunoglobulin G type antibodies, and are further described below.

  The recombinant glycoprotein of the invention can be immunoglobulin G (IgG) or comprises an IgG heavy chain. A typical IgG antibody consists of two light chains and two heavy chains that associate with each other and are further linked to two major domains: Fab fragments that are linked via a flexible hinge region. The same antigen-binding region and a heavy chain constant region capable of forming an Fc fragment are formed. The Fc region of IgG is a homodimer in which two CH1 domains are paired via non-covalent interactions. The two hinge region heavy chains between CH1 and CH2 are paired via a covalent bond. The two CH2 domains are not paired, but each has a conserved N-glycosylation site at Asn297. There are several subclasses of IgG, namely IgG1, IgG2, IgG3 and IgG4, and the recombinant glycoproteins of the present invention may belong to any one of these subclasses. The alignment of the CH2 domains of the subclass is shown in FIG. 13A, and the alignment of the heavy chain constant region is shown in FIG. 13B.

  In one embodiment of the process of the invention and the recombinant glycoprotein of the invention, the polypeptide comprises the IgG heavy chain constant segment CH2, and is defined as +3 or -1 of the N-glycosylation site as defined above. It has an amino acid residue. An example of a heavy chain constant region is shown in FIG. 13A, any of which may be used. However, preferably, the human IgG heavy chain constant segment CH2 as shown in the sequence segment of the 32 amino acid sequence segments shown at the top of FIG. 13A (lower sequence is derived from mouse). Is used. A possible alternative to the human CH2 region of FIG. 13A has a substitution of 1 to 15 amino acids, preferably 1 to 10 amino acids, based on the CH2 region (SEQ ID NO: 19) shown at the top of FIG. 13A. This is the CH2 region. Another possible alternative is a CH2 region having at least 93%, preferably at least 95%, more preferably at least 97% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 19. A further possible alternative is a CH2 region having at least 95%, preferably at least 97%, more preferably at least 99% amino acid sequence similarity to the amino acid sequence of SEQ ID NO: 19. Sequence identity was determined using Vector NTI Suite 7.1, InforMax Inc. Using the standard settings (K-tuple size: 2; best diagonal number: 4; window size 4; gap penalty 5; gap start penalty 15; gap extension penalty 6.66) Can be calculated. Amino acid sequence similarity can be determined using standard settings using BLASTX 2.2.14.

In one embodiment of the process of the invention and the recombinant glycoprotein of the invention, the polypeptide comprises an IgG heavy chain constant region and is located at the +3 or -1 position at the N-glycosylation site as defined above. Of amino acid residues. An example of a human heavy chain constant region is shown in FIG. 13B, any of which may be used. The array portion at the top of FIG. 13B is SEQ ID NO: 20. Possible alternatives to the heavy chain constant region of SEQ ID NO: 20 or the heavy chain constant region of the IGHG3 ^* 01 sequence shown in FIG. 13B are the constant region shown at the top of FIG. 13B or the IGHG3 shown in FIG. 13B. ^* A constant region having a substitution or addition of 1 to 20 amino acids, preferably 1 to 15 amino acids, more preferably 1 to 10 amino acids based on the constant region of the 01 sequence. Other possible alternatives are at least 90%, preferably at least 93%, more preferably at least 96% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 20 or the amino acid sequence of the IGHG3 ^* 01 sequence shown in FIG. 13B It is a constant region having sex. Further conceivable alternatives are at least 92%, preferably at least 95%, more preferably at least 98% amino acid sequence similarity to the amino acid sequence of SEQ ID NO: 20 or the amino acid sequence of the IGHG3 ^* 01 sequence shown in FIG. 13B Or a constant region with identity.

  In another embodiment, the polypeptide comprises an IgG heavy chain constant region (including the constant segments CH1, CH2, and CH3) and the amino acid at position +3 or −1 of the N-glycosylation site as defined above. Has a residue. In another embodiment, the polypeptide comprises an IgG heavy chain comprising a variable region and a constant region and having an amino acid residue at position +3 or −1 of an N-glycosylation site as defined above. . When the recombinant glycoprotein is or comprises an IgG heavy chain, there are no specific restrictions regarding the amino acid sequence of the variable region of the heavy chain, particularly with respect to the complementarity determining region (CDR). Suitable IgG has a binding affinity conferred by the variable region of the antibody used in the examples. Thus, a suitable IgG has a heavy chain with variable and constant regions as shown in FIG. 5 and a light chain as shown in FIG. Another suitable IgG has a heavy chain with variable and constant regions as shown in FIG. 7 and a light chain as shown in FIG.

  In the case of IgG, there is one N-glycosylation site in the heavy chain constant region, whose N-glycan occupancy can be modified according to the present invention. This N-glycosylation site is at position 297 in the Kabat sequence numbering scheme described above and shown in FIG. 1A.

  The recombinant glycoprotein may comprise a heavy chain constant region having the amino acid sequence of the constant region of human IgG, except for the amino acid at the N-glycosylation site at position +3 as defined herein.

  In one embodiment, the present invention provides a recombinant glycoprotein, which is an IgG, and a pharmaceutical composition or other composition comprising such IgG, wherein the IgG comprises the following known antibody: ibritumomab tiuxetane. , Adalimumab, cetuximab, rituximab, epatizumab, epatizumab, epatizumab, epatizumab, epatizumab, epatizumab , Abciximab, daclizumab, borociximab (Biogen Idee and PDL BioPharm), CP-675,206 (Pfizer), CAL (Roche), anti-CD80 mAb (Biogen Idee), anti-CD23 mAb (Biogen Idel), stamrumab, CAT-3888 (Cambridge Antibody Technology), CDP791 (Imclone) -X, MD1 060 (Medarex), Nimotuzumab, MDX-070 (Medarex), Matuzumab (Merck), Brentuximab, Zanolimumab, Adecatumumab, Oregovomab, Briakinumab, Denosumab, AMG108, Genmazuma, Ucrisumab, Ucrisumab , Berimumab, epratuzumab, MLN120 Has visilizumab, tocilizumab, Okurerurizumabu, certolizumab-pegol, eculizumab, pexelizumab, abciximab, Ranibijimumabu, mepolizumab, among Ibarizumabu or golimumab, the variable region of both any one identical heavy and light chains.

  Such antibodies further have both light and heavy chain constant regions as in these known antibodies, except for the modification at position +3 of the N-glycosylation site of the heavy chain constant region. Also good. Also provided are isolated nucleic acids encoding these heavy chains, nucleic acid constructs containing the nucleic acids, vectors containing the constructs, and plants containing the constructs.

  Purified IgG and their CH2 mutant variants were analyzed for glycosylation site occupancy using capillary gel electrophoresis (CGE). Glycosylated heavy chains and non-glycosylated heavy chains can be separated by molecular weight differences due to glycan addition (approximately 2-5 kDa). Electropherograms of CGE analysis of rituximab, trastuzumab and their various mutant variants are shown in FIGS. The ratio of glycosylated HC to non-glycosylated HC can be reasonably well determined by using the corresponding peak range from the electropherogram. It is necessary to load the optimal IgG onto the gel, since even larger peaks give more accurate measurements. Loading an inadequate amount of Ig from a small amount (eg, a 1 g batch) leads to inaccurate readings. However, other than this point, all the data obtained and summarized in Tables 1 and 2 convincingly demonstrate the impact on glycosylation site occupancy resulting from the substitution of various amino acid residues. Replacing the tyrosine residue with phenylalanine at position 296 has no significant effect on the glycosylation site occupancy and is neutral, while occupying the site by replacing it with glutamic acid at the same position. Cause a significant reduction in rate. In contrast, replacing the tyrosine at position 300 with threonine or glycine greatly increases the level of Fc glycosylation. Given that replacing tyrosine with phenylalanine at position 296 has no effect on glycosylation occupancy, our findings regarding IgG1 Fc can be extended to other IgG subclasses, In that case, the tyrosine at position 296 and / or 300 is replaced with phenylalanine (IgG2, IgG3, IgG4, see FIG. 13A). Interestingly, the Fc glycosylation site of anti-CD20 IgG2A antibody is occupied by more than 98% (FIG. 12B). The sequence fragment of mouse IgG2A adjacent to the N-glycosylation site is identical to one of human IgG1 except for a single amino acid substitution (YNSTLRVV vs. YNSTYRVV for mouse IgG2a and human IgG, respectively). We cannot rule out the effect of other sequence positions on the N-glycosylation site occupancy of the Fc region, including the IgG variable region sequence. However, the strongest effects on N-glycosylation levels have been demonstrated to include adjacent amino acid residues, particularly position 300. This means that replacing Y300L has a positive effect on the glycosylation site occupancy of the Fc region.

  In one embodiment, the recombinant glycoprotein is not an outer surface protein A (OspA) from a Borrelia species such as Borrelia burgdorferi, but the amino acid sequence of FIG. 1 of WO2009 / 126816 and 95 It is also not a protein with amino acid sequence identity greater than%, preferably greater than 90%, more preferably greater than 80%.

  We co-expressed trastuzumab and rituximab produced in plants together with a gene encoding the large leishmania STT3D (LmSTT3D) protein (FIGS. 1B and 2B), thereby producing an N-glycosylation site. It has been found that the occupancy of the N-glycosylation site is greatly increased without the need to introduce a mutation at position +3. The gene encoding the STT3D protein is preferably optimized for plant codons. The data shown in FIG. 12C demonstrates that the glycosylation site occupancy is similar to or slightly better than the Y300T or Y300G variants (see FIGS. 9 and 10). . Co-expression of STT3D with mutant versions of rituximab and trastuzumab (Y300T or Y300G, data not shown) shows a level of glycosylation that is indistinguishable from commercial products produced in animal cell expression systems. The usefulness of such an approach has also been demonstrated in yeast expression systems such as Pichia pastoris (Choi, BK. Et al., 2012, Appl. Microbiol. Biotechnol., 95: 671-682; US Patent Application Publication No. 2012 / 0328626).

  The present invention also provides a process for producing a recombinant glycoprotein in a plant, a plant cell and a plant cell, wherein the process comprises the step of converting a nucleic acid sequence encoding a polypeptide having an N-glycosylation site Expressing in plant cells and plant cells and co-expressing a second nucleic acid sequence encoding a heterologous single subunit oligosaccharyltransferase (OTase). The polypeptide having an N-glycosylation site may be a polypeptide described above having a particular amino acid residue at position +3, or another polypeptide having an N-glycosylation site. The second nucleic acid sequence encoding the heterologous single subunit oligosaccharyltransferase may be expressed as described above for the nucleic acid sequence of interest (first nucleic acid sequence). Expression from both viral and non-viral vectors is possible. The second nucleic acid sequence may be expressed from the same vector as the first nucleic acid sequence. In this case, the T-DNA of the binary vector may contain two expression cassettes, one used for the first nucleic acid sequence and one used for the second nucleic acid sequence. In one embodiment, both the first and second nucleic acid sequences are contained in the nucleic acid construct described above.

  In a further embodiment, a transgenic plant line containing and expressing the second nucleic acid sequence may be created, eg, expressed under the control of a constitutive or regulatory promoter. Such transgenic plants or cells thereof may then be used to express the previously described glycoproteins of the invention, eg, transiently using Agrobacterium-mediated transfection And may be expressed.

  Alternatively, the first and second nucleic acid sequences may be expressed from separate vectors. In this case, either nucleic acid sequence may be transfected or transformed into plant cells using Agrobacterium-mediated transfection or transformation as part of the T-DNA. There are two types of co-transformation: one containing a binary vector containing a first nucleic acid sequence in T-DNA and one containing a binary vector containing a second nucleic acid sequence in T-DNA. This can be easily done by mixing Agrobacterium populations or strains. The mixture may then be used for transfection, for example by infiltration or spraying as described above. Both vectors need not be either viral or non-viral vectors. For example, a first nucleic acid sequence can be expressed from a viral vector, while a second nucleic acid sequence can be expressed from a non-viral vector. The mixing ratio of the Agrobacterium population or strain may be adjusted to suit the requirements of the case. For example, if only one nucleic acid sequence is expressed from a viral vector, Agrobacterium containing a non-viral vector may be used at a higher concentration than Agrobacterium containing a viral vector.

  In a preferred embodiment, a process for producing a recombinant glycoprotein in a plant is provided, the process comprising a nucleic acid construct comprising a nucleic acid construct containing a DNA sequence of interest encoding a polypeptide having an N-glycosylation site. Transfecting a plant with a first Agrobacterium containing a first DNA molecule comprising DNA, and a second DNA sequence encoding a heterologous single subunit oligosaccharyltransferase. Transfecting the plant with a second Agrobacterium containing a second DNA molecule comprising a T-DNA containing a nucleic acid construct containing; expressing the first and second DNA sequences; Producing a glycosylated form of the polypeptide as the glycoprotein. When heterodimeric or heteromultimeric proteins are produced, the two nucleic acid sequences of interest may be expressed together with a second DNA sequence encoding a heterologous single subunit oligosaccharyltransferase. .

  For Agrobacterium that can be used to transfect the first and second DNA sequences, the information given above applies. In particular, T-DNA can be flanked by T-DNA border sequences on both sides. Either one or both of the DNA molecules may contain a selectable marker outside the T-DNA to allow cloning and genetic manipulation in bacteria. However, the T-DNA transferred to the plant cells preferably does not contain a selection marker that, if present, makes it possible to select plants or plant cells containing the T-DNA. The process preferably does not include the step of selecting plant cells in which neither the first DNA sequence nor the second DNA sequence has been incorporated using an antibiotic resistance gene or herbicide resistance gene. Therefore, neither an antibiotic resistance gene to be incorporated into the plant nor a herbicide resistance gene is required.

  The second nucleic acid sequence encodes a single subunit oligosaccharyltransferase that is heterologous to the plant (or cell) used to express it. Oligosaccharyl transferase (or “OST”) belongs to class EC 2.4.1.119. Oligosaccharyltransferase is a membrane protein complex that transfers 14-saccharide oligosaccharides from dolichol to neighboring proteins in eukaryotic cells. Oligosaccharyltransferase is a kind of glycosyltransferase. The sugar Glc3Man9GlcNAc2 (where Glc = glucose, Man = mannose, and GlcNAc = N-acetylglucosamine) is added to the asparagine (Asn) residue of the consensus sequence Asn-X-Ser or Asn-X-The; In the formula, X is any amino acid except proline. This consensus sequence is called a glycosylated sequon. The reaction catalyzed by OST is a central step in the glycosylation pathway of N-linked glycoproteins.

  The single subunit oligosaccharyltransferase can be a Leishmania protein such as STT3-A, STT3-B, STT3-C or STT3-D, or combinations thereof. The preferred source of these proteins is large leishmania. A preferred protein is the large Leishmania STT3D (LmSTT3D). The sequences of these proteins and their coding sequences are known from US 2012/0328626. The amino acid sequence of the His tag variant of LmSTT3-D expressed in the experiment reporting the results in FIG. 12C and FIG. 14 is shown in SEQ ID NO: 2 in FIG. 1B. The amino acid sequence of wild type LmSTT3-D is shown in FIG. 2D (SEQ ID NO: 22).

The present invention is not limited to any of the LmSTT3 proteins listed above and the nucleic acid sequences that encode them. Alternatively, the following nucleic acid sequence may be used as the second nucleic acid sequence:
A nucleic acid sequence encoding a protein having an amino acid sequence having at least 80%, preferably at least 90% sequence identity with the entire amino acid sequence of any one of SEQ ID NO: 2, or any one amino acid of SEQ ID NO: 2 A nucleic acid sequence encoding a protein having an amino acid sequence having at least 90%, preferably at least 95% sequence similarity with the entire sequence, or-at least 80% of the number of amino acid residues of any one of SEQ ID NO: 2, preferably A nucleic acid sequence encoding a protein having an amino acid sequence which is at least 90%, more preferably at least 95% long and has at least 90% sequence identity over such length, or-SEQ ID NO: 1 amino acid to 30 amino acids compared to any one amino acid sequence of 2 Addition of Amino Acids, nucleic acid sequence encoding a protein having an amino acid sequence having a substitution or deletion. The maximum number of amino acid additions, substitutions or deletions is at most 20, preferably at most 15, more preferably at most 10, even more preferably at most 5, whereby the total number of additions, substitutions and deletions Together determine the number of “amino acid additions, substitutions or deletions”.

  Sequence identity and similarity can be determined as described above.

  Preferably, variants of LmSTT3-D, like LmSTT3-D, increase N-glycosylation site occupancy, preferably with the same efficiency, and this occupancy is determined experimentally as described in the Examples. Can be tested. The plant cell used to express OTase preferably has its native OTase complex.

  The pharmaceutical composition of the present invention contains the recombinant glycoprotein of the present invention, preferably an IgG antibody. The pharmaceutical composition may be an aqueous liquid composition or a solid composition. Solid compositions may be prepared by lyophilization (lyophilization) from an aqueous liquid composition. The lyophilized solid composition may be reconstituted to form a liquid aqueous composition by adding water or an aqueous solvent. The aqueous liquid composition may be a composition containing a buffer and water in addition to the glycoprotein. The composition may further contain pharmaceutical excipients (see further below) such as tonicity agents and preservatives.

  In an aqueous composition, the glycoprotein is at a concentration of about 0.1 to about 10 mg / mL, preferably in an amount of about 0.5 to about 5 mg / mL, more preferably about 0.7 to 2.5 mg / mL. Can exist.

  The buffer concentration of the aqueous composition can be 5 mM to 100 mM, preferably 10 mM to 50 mM, and in one embodiment 20 mM to 30 mM. Examples of buffer substances are citric acid, maleic acid, fumaric acid, acetic acid, histidine, imidazole, organic acids and bases such as Tris and HEPES, or inorganic buffers such as phosphoric acid and diphosphoric acid. Histidine is a suitable buffer substance. The pH of the aqueous composition should be chosen to be close to human physiological pH when used for administration to a patient. The pH can be between 7.0 and 7.8. In one embodiment, the pH is between 7.2 and 7.6, more preferably 7.3 to 7.5. The pH can be set by adding an acid to the histidine aqueous solution, as is generally known. Examples of acids are inorganic acids such as hydrochloric acid and sulfuric acid, or organic acids such as citric acid, lactic acid, tartaric acid and other physiologically acceptable acids.

  The pharmaceutical composition may optionally further comprise a nonionic surfactant. Examples of surfactants are described, for example, in McCutcheon's Emulsifiers and Detergents, 1986, North American Edition, McCutcheon Division Publishing Co. , Glen Rock, New Jersey, etc. are known to those skilled in the art. For medical applications such as those used in the formulations of the present invention, pharmaceutically acceptable nonionic surfactants can be used as known to those skilled in the art. Also, combinations of nonionic surfactants can be used in the present invention.

  Of the nonionic surfactants, polysorbate surfactants are particularly preferred. Polysorbate surfactants include polysorbate 80 [poly (oxyethylene) sorbitan monooleate, also known as Tween-80], polysorbate 60 [also known as poly (oxyethylene) sorbitan monostearate, Tween-60], Polysorbate 40 [poly (oxyethylene) sorbitan monopalmitate, also known as Tween-40], polysorbate 20 [poly (oxyethylene) sorbitan monolaurate, also known as Tween-20], polysorbate 85 [poly (oxy Ethylene) sorbitan trioleate, also known as Tween-85] and polysorbate 65 [poly (oxyethylene) sorbitan tristearate, also known as Tween-65]. Those containing carboxymethyl sorbitan fatty acid esters and herein are understood. Of these, polysorbate 20 and polysorbate 80 are preferred, and polysorbate 80 is particularly preferred.

  Other nonionic surfactants that can be used in the present invention include poly (propylene oxide), and Symperonic® F-68, Pluronic® F68, Lutrol® F68, and Poloxamer 188. A block copolymer of poly (propylene oxide) and poly (ethylene oxide).

  Another additive used in the composition of the present invention that can be used in place of a nonionic surfactant is a cyclodextrin such as HP-β-CD, which may be used for protein stabilization, Can be used for parenteral administration.

  The nonionic surfactant is present in the aqueous composition or aqueous liquid formulation from about 0.001% to about 2% (w / v), preferably from about 0.01% to about 0.5% (w / v). May be present at concentrations of In a preferred embodiment, the surfactant is present at a concentration of about 0.1% (weight / volume), and in an alternative preferred embodiment is a concentration of about 0.01% (weight / volume).

  The pharmaceutical composition may contain an isotonic agent. For use in parenteral administration of pharmaceutical compositions, it is desirable to match the tonicity of the formulation to the body fluids of the human body. The tonicity agent used will be apparent to those skilled in the art. However, sugar alcohols, polyvinyl pyrrolidone and disaccharides are explicitly mentioned. Of these, mannitol and disaccharide are preferred, and disaccharide is more preferred. Particularly suitable disaccharides include trehalose and sucrose.

  The pharmaceutical composition is for adjusting buffer, antioxidant, bacteriostatic agent, diluent, carrier, surfactant, salt, preservative, stabilizer, wetting or solubilizing agent, and / or osmotic pressure. It may further contain one or more additional pharmaceutically acceptable excipients such as

  In general, a pharmaceutical composition herein means a composition suitable for administration to a patient, particularly a human patient. The pharmaceutical composition may be administered to a patient by a parenteral administration technique, preferably by subcutaneous or intramuscular administration, more preferably by a subcutaneous administration technique.

  The pharmaceutical composition or glycoprotein of the present invention can be used in a therapeutic method. Preferably, it is used for the treatment of cancer. Examples of cancer are Her2 / Neu positive cancers as listed below.

The human epidermal growth factor receptor Her2, also known as Neu, ErbB-2 or p185, is a member of the epidermal growth factor receptor (EGFR / ErbB) family and is encoded by the ERBB2 gene. Like other members of the ErbB family, Her2 is a membrane-bound receptor tyrosine kinase that consists of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain that can interact with downstream signal molecules. ERBB2 gene amplification occurs in 20-30% of human breast and ovarian cancers and is linked to a more aggressive disease course and worse prognosis (Bange, J., Zwick E. and Ullrich A). , 2001, Nature Medicine, 7: 548-552; Slamon, DJ, Clark, GM, Wong, SG, et al., 1987 'Science, 235: 177-182; , Godolphin, W., Jones, LA et al., 1989, Science, 244: 707-712; Berchuck, A., Kamel, A., Whitaker.R. Et al., 1990, Cancer Research, 50: 4087- 4091). In ERBB2 ⁺ tumor cells, the receptor functions heterologously with another ErbB member to function itself and / or transmit an uncontrolled proliferation signal that is responsible for the neoplastic behavior of the cell. It is necessary to form a dimer. HER2 has been developed as an important target for breast cancer treatment, particularly by monoclonal antibody therapy. For example, Herceptin (trastuzumab), a humanized monoclonal antibody against this surface target, was approved by the FDA in 1998. Herceptin significantly affects the survival of HER2-positive breast cancer patients (Tan, AR and Swain, SM, 2002, Seminars in Oncology, 30: 54-64). Trastuzumab has activity against HER2 homodimers but is not effective against ligand-induced HER2 heterodimers (Agus, DB, Akita, RW, Fox, WD). Et al., 2002, Cancer Cell, 2: 127-137; Cho, HS, Mason, K., Ramyar, KX, et al., 2003, Nature, 421: 756-60). Trastuzumab is effective in treating late stage metastatic cancer. US Pat. Nos. 7,449,184 and 7,981,418 describe antibody therapy for HER2 / neu positive metastatic breast cancer.

  In order to treat Her2 / Neu positive cancer, the employed glycoprotein may have an affinity with Her2 / Neu. Preferably, for this purpose, the glycoprotein is an antibody having affinity for HER2 / Neu, such as an antibody having trastuzumab heavy and light chain variable domains.

  The following examples further illustrate the present invention. However, the present invention is not limited to these examples.

(Example 1)
Design binary vectors for constructs are described in the MAGICON® technology (Gleba, Y., Klimyuk, V. and Marilonnet, S., 2005, Vaccine 23: 2042-2048; Gleba, Y., Klimyuk, V. and Marilonnet, S., 2007, Curr. Opin.Biotechnol., 18: 134-141), elements derived from tobacco mosaic virus (TMV) or potato virus X (PVX) (Giritch et al., 2006, Proc. Natl. Acad. Sci. USA, 103: 14701-14706), developed at Icon Genetics.

  TMV vectors are two closely related plant viruses that naturally infect Solanum and Brassicaceae plants by transmission by physical forces, namely TVCV (Turn Vein Permeabilizing Virus, Lartey, RT, Lane). , LC and Melcher, U., 1994, Arch. Virol., 138: 287-298; Latey, RT, Voss, TC and Melcher, U., 1995, Gene, 166: 331. 332) and crTMV (constructed based on the cDNA of rape infectious tobamovirus, Dorokhov, YL, Ivanov, PA, Novikov, VK, et al., 1994, FEBS Lett., 350: 5-8). did. Since both parental viruses are tobamoviruses and are related to the well-known tobacco mosaic virus (TMV), we refer to the resulting vector as “TMV-based”. All three viruses (TVCV, crTMV and TMV) are positive-strand RNA viruses and have the same overall structure and mode of replication. Basically, these viruses express the following proteins: (1) its function is to express the complete viral RNA transcript (genomic RNA), as well as two other viral proteins (migrating protein and coat protein). RNA-dependent RNA polymerase (RdRp), which replicates two types of subgenomic RNA (sgRNA) required for (2), and (2) intercellular movement of viral genomic RNA (short-range movement limited to infiltrated leaves) And (3) a coat protein (CP) required for the formation of virus particles and movement throughout the body (long-distance movement from leaf to leaf via the vascular system). Code. Interestingly, virus particle formation is not necessary for cell-to-cell movement. Therefore, the present inventors have removed the coat protein from their vector and replaced it with the target gene. This has two consequences: the viral vector cannot make virus particles, and the gene of interest is expressed at a higher level than if the viral vector contains both CP and the gene of interest, Has been brought. In addition to the viral protein and the gene of interest, the viral vector should also contain 5 ′ and 3 ′ untranslated (5′ntr and 3′ntr) viral sequences (Malllonnet, S., Giritch, A., Gils, M. et al., 2004, Proc. Natl. Acad. Sci. USA, 101: 6852-6857). In order to efficiently express TMV-based viral vectors in plant cells, the viral vector cDNAs have been cloned between plant promoters and plant terminators (Act2 and nos) (Malllonnet, S., Giritch, A. et al. Gils, M. et al., 2004, Proc. Natl. Acad. Sci. USA., 101: 6852-6857), plant introns were added within the RdRP sequence and within the MP sequence (Malllonnet, S., Toeringer, C., Kandsia, R. et al., 2005, Nat. Biotechnol., 23: 718-723).

  In order to express a heteromultimeric protein such as an immunoglobulin, it is necessary to express the two proteins in the same cell, in this case the immunoglobulin heavy and light chains. Since two different viral vectors based on TMV tend to exclude each other in infected cells (only one vector will succeed in replication in one cell), we Two different viral vectors constructed on the cDNA of two viruses capable of co-replication in infected cells were used. This second virus is potato virus X [PVX; isolated PV-0018, “Deutsches Sammlung von Mikroorganisund und Zellkulturen GmbH”, accession number PV-0018]. PVX is a positive-strand RNA virus of the family Flexiviridae Potexvirus that naturally infects solanaceous and cruciferous plants by transmission through physical forces. PVX has a different mode of replication than TMV. Like TMV, PVX is an RdRp for replication, 3 genes for cell-to-cell movement (3 gene blocks consisting of 25K, 12K and 8K proteins), and the formation of viral particles and systemic migration For containing CP. An important difference from TMV is that PVCP is also required for cell-to-cell movement. Therefore, CP cannot be removed from the viral vector. In order to obtain a high level of expression, the gene of interest is cloned after the CP gene. In contrast to TMV-based vectors, introns are not required to efficiently transport the original PVX vector transcript from the nucleus to the cytosol.

  For TMV vectors we use a promoter that is active in plants to generate the initial transcript. However, in the case of PVX vectors, we use a constant 35S promoter derived from cauliflower mosaic virus. Plant terminators are not required for vectors based on PVX. Finally, we have used Agrobacterium tumefaciens for both TMV-based and PVX-based vectors to efficiently deliver viral vectors to plant cells. Therefore, a complete viral vector (plant promoter for TMV-based vector, viral vector sequence with the gene of interest, and plant terminator) was cloned between the left and right borders of the binary vector T-DNA. (FIG. 3). The elements of the binary vector are: (1) pVS origin of replication for replicating the plasmid in Agrobacterium (Hajdukiwicz, P., Svab, Z. and Maliga, P., 1994, Plant Mol Biol., 25: 989-994), (2) colE1 origin for replicating the plasmid to a high level in E. coli, (3) antibiotic resistance gene (nptIII conferring kanamycin resistance, Frisch, DA, Harris) -Haller, L.W., Yokubatis, N.T., et al., 1995, Plant Mol. Biol., 27: 405-409), and (4) clearly defining the ends of the DNA pieces transferred to the plant cells. T-DNA left and right borders (Frisc , D.A., Harris-Haller, L.W., Yokubaitis, N.T et al., 1995, Plant Mol.Biol, 27:.. 405~409) is. To facilitate blue / white selection, a lacZα cassette amplified from pUC19 was inserted between two BsaI restriction enzyme sites that allowed the gene of interest to be cloned in frame seamlessly. Therefore, during the initial construction of the viral vector, all naturally occurring BsaI recognition sites were removed so that the target gene could be cloned easily and robustly.

  Based on the amino acid sequences of trastuzumab and rituximab, variable region versions optimized for tobacco codons were synthesized (FIGS. 4-7). In addition, universal gamma 1 heavy chain and kappa light chain constant regions were synthesized using tobacco codon usage (FIG. 1A, FIGS. 4-7). A BsaI restriction enzyme site of the type IIS enzyme was added to both ends of the sequence to generate a DNA module that could be cloned into a plant virus expression vector based on magnICON® (FIG. 2A, FIG. 3). . In the universal gamma 1 heavy chain constant region of trastuzumab and rituximab, six single mutations (E293R, E294Y, Y296F, Y296E, Y300T and Y300G) and one triple mutation (E294L + Y296T + Y300T) (FIG. 1A) were introduced by PCR. It was. The resulting gamma 1 constant region DNA module was cloned into the PVX and TMV based magnICON® plant virus vector using the IIS type enzyme BsaI, together with the signal peptide and variable region sequences ( FIG. 2A, FIG. 3). FIG. 2 depicts the cloning of fragments with mutations that result in regulation of IgG Fc glycosylation. The resulting heavy chain construct was combined with the respective light chain construct used for plant expression.

  Cloning of the large Leishmania STT3-D gene (LmJF35.1160, accession number E9AET9) was performed in the same manner as the cloning of the IgG1 heavy chain and light chain. A codon-optimized version of corn based on the amino acid sequence of large Leishmania STT3-D (LmJF35.1160, accession number E9AET9) with a His tag at the C-terminus and adjacent to a BsaI type IIS restriction enzyme site Was synthesized (FIG. 1B). A BsaI restriction enzyme site was added to generate a DNA module that could be cloned between the 35S promoter and OCS terminator together seamlessly into a binary expression vector (FIG. 2B).

(Example 2)
Expression, Purification and Glycosylation Analysis of Recombinant Plant Produced Antibodies with Rituximab Variable Regions Both Agrobacterium strains (Giritch et al., 2006) carrying PVX and TMV vectors to produce Ig Proc. Natl. Acad. Sci. USA, 103: 14701-14706) were grown separately in LB medium in which tryptone was replaced with soy peptone (Duchefa Biochemie, Haarlem, The Netherlands). Bacterial cultures were grown at 28 ° C. until OD ₆₀₀ reached 3-4. Both bacterial cultures are mixed with invasion buffer (10 mM MES, pH 5.5, 10 mM MgSO4) to achieve a defined cell density (equivalent to a 500-fold dilution of a culture with an OD _{600 of} 1.0). An infiltration solution was prepared by dilution. Bensamiana tobacco plants grown under controlled and standardized conditions are infiltrated under reduced pressure using an Agrobacterium suspension (a mixture of two Agrobacterium strains carrying TMV and PVX vectors), Subsequently, it was kept in the growth chamber for 7 days, and Ig was expressed and accumulated. The plant biomass is then harvested (5 kg per batch) and mixed with 2 volumes (weight / volume) of extraction buffer (200 mM Tris-HCl, pH 7.5, 5 mM EDTA) pre-cooled to + 4 ° C. in a blender Homogenized with. The pH of the homogenate was lowered to <5.1 to remove host cell proteins including rubisco. Subsequently, the pH of the homogenate was adjusted to 8.5 using NaOH and then the crude extract was clarified by dead end filtration. Ig was purified from the clarified extract using protein A affinity chromatography. The affinity eluate was ultra / diafiltered against PBS (10 mM NaH 2 PO 4, 137 mM NaCl, pH 7.3) and a final protein concentration of 0.5-1.5 mg / mL using PBS buffer. Adjusted. Small scale green biomass (1 g, 100 g, up to 1 kg) was processed as described in Example 3.

  Capillary gel electrophoresis (CGE) was used to determine the N-glycan occupancy of the antibody. For CGE analysis, an Agilent bioanalyzer and Protein 230 kit were used according to the manufacturer's instructions. For analysis, 4 μL of protein sample was combined with 2 μL of reduced sample buffer and denatured sample buffer. To estimate the protein size, the electrophoretic mobility of the protein in the sample was compared to the mobility of a molecular weight marker (a mixture of proteins of various sizes with a given mass).

  Electrophorograms of reduced plant-produced rituximab and its Fc variant are shown in FIGS. 8, 9, and 12. FIG. The heavy chain of plant-produced antibodies is divided into two peaks with molecular weights of 53.4 kDa and 58.9 kDa, corresponding to the unglycosylated and glycosylated forms, respectively. This was confirmed by mass spectral analysis of the glycopeptide (Example 4). The N-glycosylation site occupancy of the heavy chain of rituximab expressed by plants is approximately 79% for wild type Fc. This number dropped to 5-6% for the Y300T and Y300G Fc variants. Table 1 summarizes the N-glycosylation site occupancy of various Fc variants of plant-produced rituximab.

In addition, plant-produced rituximab and its Fc mutant variant were co-expressed together with the STT3D gene. The process of performing the experiments, including plant infiltration, incubation, and IgG purification, was performed in the same manner as previously described, except that agro with the STT3D expression cassette was added to the infiltration mixture. However, considering that STT3D is expressed from a non-viral vector (pICH94911 depicted in FIG. 2B), the concentration of Agrobacterium carrying T-DNA with an expression cassette for STT3D is About 50 times higher. The concentration of Agrobacterium containing the vector was equivalent to a 4-fold dilution of a culture with an OD ₆₀₀ of 1.0. The results are shown in FIG. 12C.

Table 1. Percentage of non-glycosylated IgGl Fc in various production batches of plant produced rituximab. The range size of the corresponding HC peak from the capillary gel electrophoresis scan was used to calculate a measure of the ratio of glycosylated heavy chain and non-glycosylated heavy chain (“HC glyc” and “HC aglyc”, respectively). CHO-CHO cell culture; b. wt-benzamiana tobacco (wild-type) plant; b. N. benthamiana tobacco plants having genes silenced fucosyltransferase and xylosyltransferase by RNAi-RNAi (Strasser, R. et al., 2008, Plant Biotechnol. J., 6: 392-402); b. RNAi (ΔXylT / FucT) —a knockout gene (chemical mutation of 2 genes encoding xylosyltransferase and 5 genes encoding fucosyltransferase) and RNAi silencing for fucosyltransferase and xylosyltransferase Tobacco plants; MabThera—commercially available rituximab; RIT-plant-made rituximab; Y296F, E294Y, Y300T, E294L-Y296T-Y300T, etc.—variants of rituximab produced in plants, corresponding to amino acid substitutions in the Fc region The name of the variant.

(Example 3)
Expression, purification and glycosylation analysis of recombinant plant-produced trastuzumab To produce recombinant protein, tryptone was replaced with soybean peptone (Duchefa Biochemie, Haarlem, The Netherlands) and 50 μg / mL rifampicin and 50 μg / mL kanamycin Selected Agrobacterium strains carrying TMV-based expression vectors were grown in supplemented LBS liquid medium. Agrobacterium cultures were grown at 28 ° C. until OD ₆₀₀ reached 2-4. Dilute the Agrobacterium culture in invasion buffer (10 mM MES, pH 5.5, 10 mM MgSO4) to a defined cell density (equivalent to a 200-fold dilution of a culture with an OD _{600 of} 1.0). The infiltration solution was prepared by

  About 4 to 10 Bensamiana tobacco plants grown for 6-8 weeks under controlled and standardized conditions are vacuum infiltrated with Agrobacterium infiltration solution and then placed in the greenhouse for 6-8 days The recombinant protein was expressed and accumulated. The plant leaves were then harvested and ground in liquid nitrogen to a fine leaf powder and kept at −80 ° C. until protein extraction and subsequent purification.

  Leaf powder (batch size from 1 g to 1 kg) was extracted in about twice the amount (weight / volume) of extraction buffer (20 mM sodium phosphate, pH 6.0, 0.5 M NaCl). Extraction was carried out on a stirrer at + 4 ° C. for 40 minutes. The homogenate was clarified by centrifugation at 15,000 × g for 10 minutes, followed by filtration through a MiraClot filter. The pelleted plant tissue was re-extracted using the same extraction conditions. The extracts were combined and subjected to pH adjustment. The pH of the clarified homogenate was lowered to 5.0 using 5N HCl to remove host cell proteins including rubisco. After incubation for about 30 minutes with stirring at pH 5, the pH of the crude extract was readjusted to pH 7.4 using 5N NaOH. The crude extract was then centrifuged (20,000 × g for 15 minutes) to remove cell debris and sediment. Prior to applying the crude extract to the affinity chromatography column, the crude extract was filtered through several filtration membranes (20 μm-8 μm-3 μm-0.45 μm). The clear filtrate was applied onto a spin column filled with 100 μL protein A sepharose (GE Healthcare, 17-5255-01) and equilibrated with 10 CV wash buffer (20 mM sodium phosphate, pH 7.3). . After loading, the column was washed with 20 CV (column volume) wash buffer. The antibody was eluted using 5 CV elution buffer (10 mM sodium phosphate pH 3.0, 137 mM NaCl).

  The eluted IgG was ultra / diafiltered using a Spin-X UF concentrator 30k MWCO (Corning, # 431489). Finally, the concentrate was sterile filtered using a 0.2 μm filter. Using the protocol described in Example 2, a batch of 5 kg of green biomass was processed. Antibody N-glycan occupancy was determined as described in Example 2.

  Electropherograms of reduced plant-produced trastuzumab and its Fc variant version are shown in FIGS. The heavy chain (HC) of plant-produced trastuzumab is separated into two peaks with molecular weights of approximately 53.4 kDa and 58.9 kDa. The 53.4 kDa peak corresponds to the non-glycosylated heavy chain variant. Table 2 summarizes the N-glycosylation site occupancy of various Fc variants of plant-produced trastuzumab.

In addition, plant-produced trastuzumab and its Fc mutant variant were co-expressed together with the STT3D gene. The process of performing the experiment, including plant invasion, incubation, and IgG purification, is preceded by the addition of Agrobacterium with the STT3D expression cassette in T-DNA to the Agrobacterium mixture for infiltration. Performed in the same manner as described. However, considering that STT3D is expressed from a non-viral vector, the concentration of Agrobacterium carrying T-DNA having an expression cassette for STT3D was approximately 50 times higher than that of the viral vector. The concentration of Agrobacterium containing the vector was equivalent to a 4-fold dilution of a culture with an OD ₆₀₀ of 1. The results are shown in FIG. 12C.

Table 2. Percentage of non-glycosylated IgG1 Fc in various production batches of plant-produced trastuzumab. The range size of the corresponding HC peak from the capillary gel electrophoresis scan was used to calculate a measure of the ratio of glycosylated heavy chain and non-glycosylated heavy chain (HC glyc and HC aglyc, respectively). CHO-CHO cell culture; b. wt-benzamiana tobacco (wild-type) plant; b. Bensamiana tobacco plant with genes knocked down for RNAi-fucosyltransferase and xylosyltransferase (Strasser, R., et al., 2008, Plant Biotechnol. J., 6: 392-402); Herceptin—commercially available trastuzumab; TRA -Trastuzumab produced in plants; Y296F, E294Y, E294L-Y296T-Y300T, etc.-Trastuzumab variants produced in plants, the names of variants corresponding to amino acid substitutions in the Fc region.

(Example 4)
Analysis of glycopeptide produced by trypsin treatment by reversed-phase LC-ESI-MS 10 μg of each sample was S-alkylated using iodoacetamide. Protein was recovered by precipitation with acetone and digested with sequencing grade trypsin (Roche). Approximately 3 μg of each digest was loaded onto a BioBasic C18 column (BioBasic-18, 150 × 0.32 mm, 5 μm, Thermo Scientific) using 65 mM ammonium formate at pH 3.0 as the aqueous solvent. A gradient of 10% to 55% acetonitrile was developed over 20 minutes at a flow rate of 6 μL / min. Detection was carried out in positive ion and simple MS mode using a Q-TOF mass spectrometer (Waters Micromass Q-TOF Ultimate Global).

MS profiles were set to 450, 750 and 950. The instrument was calibrated using cesium iodide. Glycopeptides were identified by oligosaccharide analysis as a set of peaks consisting of peptide moieties and added N-glycans with various sugar residues (Man, GlcNAc, Gal or Fuc) added. The theoretical masses of these glycopeptides were determined using a spreadsheet using monoisotopic masses for amino acids and monosaccharides. A descriptive integration of the totaled spectrum with MaxEnt3 gave a picture of the glycosylation state at each site. The quantitative presence of each glycopeptide mass variant was estimated from the peak height of the MaxEnt3 spectrum. The hypothetical average mass of glycans was calculated as the sum of the weighted average (with additional mass due to sugar). Thus, non-glycosylated variants (such as those in the Fc region) have a “glycan mass” of zero. As an example, the calculated percentages of unglycosylated Fc glycopeptides and glycosylated Fc glycopeptides of various glycoforms are shown in Table 3.

Table 3. Quantification of the total percentage (%) of plant-produced trastuzumab glycopeptides in which the heavy chain N-glycosylation site is not modified

  The content of European Patent Application No. 13002866.5 filed on June 4, 2013 is incorporated herein, including the description, claims and drawings.

Claims (22)

  A process for producing a recombinant glycoprotein in a plant, plant cell or plant cell, wherein a nucleic acid sequence encoding a polypeptide having an N-glycosylation site is expressed in said plant, said plant cell or said plant cell And co-expressing a nucleic acid sequence encoding a heterologous single subunit oligosaccharyltransferase (OTase).   A process for producing a recombinant glycoprotein in a plant, comprising a first DNA molecule comprising a T-DNA comprising a nucleic acid construct comprising a DNA sequence of interest encoding a polypeptide having an N-glycosylation site A nucleic acid construct containing a second DNA sequence encoding a heterologous single subunit oligosaccharyltransferase (OTase) using a first Agrobacterium to transfect said plant Transfecting the plant with a second Agrobacterium containing a second DNA molecule comprising a T-DNA comprising: expressing the first and second DNA sequences; Glycosylated form of the polypeptide as the glycoprotein And a step of raw, the process.   A single subunit OTase is class EC. Process according to claim 1 or 2, which belongs to 2.4.1.119 and transfers the oligosaccharide Glc3Man9GlcNAc2 from doricol to an asparagine residue at the N-glycosylation site of the nascent protein in eukaryotic cells.   4. The process of any one of claims 1-3, wherein the single subunit OTase is a Leishmania STT3A protein, STT3B protein, STT3C protein, STT3D protein, or a combination thereof. A nucleic acid sequence encoding a single subunit OTase is
i. A nucleic acid sequence encoding a protein having an amino acid sequence having at least 80%, preferably at least 90% sequence identity with the entire amino acid sequence of any one of SEQ ID NO: 2, or ii. A nucleic acid sequence encoding a protein having an amino acid sequence having at least 90%, preferably at least 95% sequence similarity with the entire amino acid sequence of any one of SEQ ID NO: 2, or iii. At least 80%, preferably at least 90%, more preferably at least 95% of the number of amino acid residues of any one of SEQ ID NO: 2 and at least 90% of the sequence over such length A nucleic acid sequence encoding a protein having an amino acid sequence with identity, or iv. A nucleic acid sequence encoding or comprising a protein having an amino acid sequence having 1 to 30 amino acid additions, substitutions or deletions compared to any one amino acid sequence of SEQ ID NO: 2 The process according to any one of up to. A process for producing a recombinant glycoprotein in a plant, plant cell or plant cell comprising the step of expressing a nucleic acid sequence encoding a polypeptide in said plant, said plant cell or said plant cell Has an N-glycosylation site of the consensus sequence Asn-X-Ser or Asn-X-Thr, where X is any standard amino acid residue, where Asn of said N-glycosylation site When the residue is assigned to position 0 of the amino acid sequence,
(A) the amino acid residue at position +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met, or (b) the amino acid residue at position -1 is selected from Glu and Asp ,
The above process. A nucleic acid sequence encoding a polypeptide comprising CH2 of an IgG heavy chain constant segment having the N-glycosylation site of the consensus sequence Asn-X-Ser or Asn-X-Thr is obtained in the plant, the plant cell or the plant cell Wherein X is any standard amino acid residue, wherein the Asn residue of the N-glycosylation site is assigned to position 0 of the amino acid sequence,
(A) the amino acid residue at position +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met, or (b) the amino acid residue at position -1 is selected from Glu and Asp ,
The process of claim 6.   The process according to claim 6 or 7, wherein the amino acid residue at the +3 position is Thr.   Said polypeptide encoded by said nucleic acid sequence comprises an IgG heavy chain constant region, preferably said polypeptide is or comprises an immunoglobulin G (IgG) heavy chain, preferably by said nucleic acid sequence 9. The polypeptide according to any one of claims 6 to 8, wherein the encoded polypeptide is a human immunoglobulin G (IgG) heavy chain except for the amino acid residues at positions +3 and -1 according to claim 1. The process described in   Co-expressing a second nucleic acid sequence encoding a heterologous single subunit oligosaccharyltransferase in said plant, plant cell or plant cell, wherein said nucleic acid sequence encoding a polypeptide and said 10. Process according to any one of claims 6 to 9, wherein two nucleic acid sequences can both be transiently expressed in the plant, preferably by transfection mediated by Agrobacterium.   11. A process according to any one of claims 6 to 10, wherein the recombinant glycoprotein is not an outer surface protein A (OspA) from a Borrelia species such as Borrelia burgdorferi. .   The plant is a higher plant, preferably a dicotyledonous plant, more preferably a Solanasacea plant, more preferably a Nicotiana plant, most preferably Nicotiana benthamiana, and the plant cell Is a cell derived from a higher plant, preferably a dicotyledonous plant, more preferably a Solanasae plant, more preferably a Nicotiana plant, most preferably Nicotiana benthamiana. The process according to any one of 1 to 11. A process for regulating glycosylation occupancy of a N-glycosylation site of a polypeptide after expression in a plant or plant cell, said polypeptide having the consensus sequence Asn-X-Ser or Asn-X-Thr Comprising the IgG heavy chain constant segment CH2 with said N-glycosylation site, X is any standard amino acid residue;
A nucleic acid sequence encoding a polypeptide, wherein a codon encoding a Tyr or Phe residue at position +3 counting from the Asn residue at position 0 of the N-glycosylation site is defined as Thr, Ser, Leu, Ile, Val and Met. Substituting a codon encoding an amino acid residue selected from:
Expressing the nucleic acid sequence having the substitution made in the plant or the plant cell in the previous step.   A recombinant glycoprotein produced or capable of being produced according to any one of claims 1-13. A recombinant glycoprotein comprising a polypeptide comprising CH2 of an IgG heavy chain constant segment having a glycosylated N-glycosylation site of the consensus sequence Asn-X-Ser or Asn-X-Thr, wherein X is any Wherein the Asn residue of the N-glycosylation site is assigned to position 0 of the amino acid sequence,
(A) the amino acid residue at position +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met; or (b) the amino acid residue at position -1 is selected from Glu and Asp;
The above recombinant glycoprotein, which may be a glycoprotein expressed in a plant or plant cell.   The recombinant glycoprotein of item (a) has a glycosylation occupancy at the N-glycosylation site of at least 85%, more preferably at least 90%, or the glycoprotein of item (b) 16. A recombinant glycoprotein according to claim 15, having a glycosylation occupancy at the N-glycosylation site of at most 65%, preferably at most 55%.   The polypeptide comprises an IgG heavy chain constant region, or the polypeptide comprises an IgG heavy chain comprising a variable region and a constant region, preferably the polypeptide comprises the +3 position or-of claim 1 The human IgG heavy chain excluding the amino acid residue at position 1, or the recombinant glycoprotein is an IgG antibody or an Fc fragment of an IgG antibody according to any one of claims 14 to 16. Recombinant glycoprotein.   A pharmaceutical composition comprising the recombinant glycoprotein according to any one of claims 14 to 17 and a pharmaceutically acceptable carrier.   18. A recombinant glycoprotein according to any one of claims 14 to 17 for use in therapy, for treating cancer, or for treating Her2 / Neu positive cancer in a patient. An isolated nucleic acid encoding a polypeptide comprising CH2 of an IgG heavy chain constant segment with the N-glycosylation site of the consensus sequence Asn-X-Ser, wherein X is any standard amino acid residue Yes, where the Asn residue of the N-glycosylation site is assigned to position 0 of the amino acid sequence,
(A) the amino acid residue at position +3 is selected from Thr, Ser, Gly, Leu, Ile, Val and Met, or (b) the amino acid residue at position -1 is selected from Glu and Asp ,
The isolated nucleic acid.   A plant or plant cell comprising a higher plant or higher plant cell comprising a nucleic acid construct comprising the nucleic acid construct of claim 20, or a vector comprising the nucleic acid construct.   A higher plant or higher plant cell comprising a nucleic acid sequence encoding a heterologous single subunit oligosaccharyltransferase.